P2X3 and/or P2X2/3 compounds and methods

ABSTRACT

The present disclosure provides novel compounds and methods for preparing and using these compounds. In one embodiment, the compounds are of the structure of formula (I), wherein R1-R7 are defined herein. In a further embodiment, these compounds are useful in method for regulating one or both of the P2X3 or P2X2/3 receptors. In another embodiment, these compounds are useful for treating pain in patients by administering one or more of the compounds to a patient. In another embodiment, these compounds are useful for treating respiratory dysfunction in patients by administering one or more of the compounds to a patient.

TECHNICAL FIELD

The present disclosure relates to compounds and methods for regulatingactivity of one or both of the P2X₃ or P2X_(2/3) receptors.

BACKGROUND

Adenosine triphosphate (ATP) is well-recognized as the primary energycurrency of living cells, but has also emerged as a significantsignaling molecule that can shape physiological and pathophysiologicalprocesses by interacting with any of several ‘purinergic’membrane-associated receptor molecules. The purinergic receptor familycomprises both G-protein-coupled (GPCR) receptors (assigned a “P2Y”nomenclature) and ligand-gated ion channel or “P2X” variants.

ATP elicits an excitatory effect on afferent sensory nerves via aninteraction with receptors of the P2X subfamily. The consequence of suchhyperexcitability can be interpreted as pain when the ATP effect iselicited in skin, bone or visceral tissues, as pain and/or cough inairway tissues, or as pain and/or instability when it occurs in thebladder. See, e.g., Ford, Purinergic Signalling, 8 (Suppl 1), S3-S26,2012 and Ford et al., Frontiers in Cellular Neuroscience, Volume 7,Article 267, 2013. Two particular receptor variants within the P2Xsubfamily, designated P2X₃ and P2X_(2/3), have emerged as targets ofparticular interest in a variety of studies designed to measurenociception, airway or bladder function in rodents, since activation ofthese receptors by ATP is capable of generating the adverse events citedabove. Agents which target P2X subfamily receptors would therefore havetherapeutic value in treating conditions associated with thesereceptors, for example pain, respiratory disorders or bladderdysfunction.

Accordingly, there is a need for more potent and selectiveP2X₃/P2X_(2/3) modulators.

SUMMARY

In one embodiment, a compound of formula (I) is provided, wherein R¹-R⁷are as described herein, or a pharmaceutically acceptable salt thereof.

In another embodiment, the present disclosure provides a pharmaceuticalcomposition comprising a compound of formula (I) and a pharmaceuticallyacceptable excipient.

In another embodiment, the present disclosure provides a kit comprisinga compound of formula (I) or a pharmaceutical composition comprising acompound of formula (I).

In another embodiment, the present disclosure provides a method forregulating activity of one or both of the P2X₃ or P2X_(2/3) receptors,comprising administering a compound of formula (I) to a subject in needthereof. In certain embodiments, regulating activity of one or both ofthe P2X₃ or P2X_(2/3) receptors comprises inhibition of those receptors.

In another embodiment, the present disclosure provides a method formodulating one or both of the P2X₃ or P2X_(2/3) pathways, comprisingadministering a compound of formula (I) to a patient.

In another embodiment, the present disclosure provides a method fortreating pain in a subject, comprising administering to the subject atherapeutically effective amount of a compound of formula (I) or apharmaceutical composition comprising a compound of formula (I).

In another embodiment, the present disclosure provides a method fortreating a respiratory dysfunction in a subject, comprisingadministering to the subject a therapeutically effective amount of acompound of formula (I) or a pharmaceutical composition comprising acompound of formula (I). In some embodiments, the respiratorydysfunction is cough.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a map of a circular cloning plasmid pIRESpuro3 (ClontechLaboratories Inc., Mountain View, Calif.) into which the protein codingsequence of rat P2X2 has been cloned in the EcoRV-digested anddephosphorylated vector pIRES-puro3 within a multiple cloning site(MCS).

FIG. 1B is a map of a circular plasmid pCDNA-Hygro (Invitrogen) intowhich the coding sequence of rat P2X3 (NCBI Accession No: X91167),amplified by PCR from rat brain cDNA, has been cloned. The PCR productobtained containing the protein coding sequence of rat P2X3 was clonedinto EcoRV-digested and dephosphorylated vector pCDNA-Hygro within themultiple cloning site (MCS).

FIG. 1C is a vector generated from the pcDNA-Hygro containing the ratP2X3, which was then subcloned into pcDNA-5/TO (Invitrogen/LifeTechnologies) at HindII (5′) and XhoI (3′) sites within the multiplecloning site (MCS) of the vector.

DETAILED DESCRIPTION

Discussed herein are novel compounds which can modulate the activity ofone or both of the P2X₃ or P2X_(2/3) pathways. These compounds can beused to treat any disease or disorder affected by a dysregulation of oneor both of the P2X₃ or P2X_(2/3) pathways.

The present disclosure thus provides compounds having the structure offormula (I), or prodrugs, enantiomers or pharmaceutically acceptablesalts thereof:

R¹ is H, halogen, C₁-C₆ alkyl, C₃-C₆ cycloalkyl, CN or CONH₂;

R² is none, C₁-C₆ alkyl or aryl;

R³ is none, optionally substituted C₁-C₆ alkyl, C₃-C₆ cycloalkyl orheterocycle;

R⁴ is optionally substituted heteroaryl, optionally substituted C₁-C₆alkyl, optionally substituted C₃-C₆ cycloalkyl or optionally substitutedaryl;

R⁵, R⁶ are independently H, C₁-C₆ alkyl or C₃-C₆ cycloalkyl; and

R⁷ is CONHR⁸, optionally substituted heterocycle or optionallysubstituted heteroaryl, wherein R₈ is H, alkyl or cycloalkyl.

In some embodiments, the optional substituents for R³, R⁴ and R⁷ arehalogen, C₁-C₆ alkyl, C₃-C₆ cycloalkyl, CONH₂, C₁-C₆ alkoxy optionallysubstituted with halogen or C₃-C₆ cycloalkyl, heterocycle, heteroaryl oraryl optionally substituted with halogen or C₃-C₆ cycloalkyl.

In certain embodiments, compounds of formula (I) are provided wherein R¹is H.

In certain embodiments, compounds of formula (I) are provided wherein R²is optionally substituted C₁-C₆ alkyl.

In certain embodiments, compounds of formula (I) are provided wherein R³is H.

In certain embodiments, compounds of formula (I) are provided wherein R¹is halogen, C₁-C₆ alkyl, or CN.

In certain embodiments, compounds of formula (I) are provided wherein R⁷is CONHR⁸, wherein R⁸ is H, alkyl or cycloalkyl. In some embodiments, R⁸is cyclopropyl.

In certain embodiments, compounds of formula (I) are provided wherein R⁷is an optionally substituted morpholine or piperazine, for example

wherein X is O or H, H; and R⁹ is H, optionally substituted C₁-C₆ alkyl,CO(C₁-C₆ alkyl), CONH₂, C(NH)NH₂, C(N—CN)NH₂ or SO₂NH₂.

In certain embodiments, compounds of formula (I) are provided wherein R⁷is an optionally substituted heteroaryl selected from the groupconsisting of pyrazole, triazole, oxadiazole or pyridazine, for example:

wherein R¹⁰ is H or alkyl optionally substituted with —OH; R¹¹ is H,CH₂—CONH₂ or alkyl optionally substituted with —OH or halogen; and R¹²is H or CONH₂.

In certain embodiments, compounds of formula (I) are provided wherein R⁹is CO(C₁-C₆ alkyl) or CONH₂.

In certain embodiments, compounds of formula (I) are provided wherein R⁴is optionally substituted heteroaryl.

In certain embodiments, compounds of formula (I) are provided wherein R⁴is optionally substituted aryl.

In certain embodiments, compounds of formula (I) are provided wherein R¹and R³ are H.

In certain embodiments, compounds of formula (I) are provided wherein R⁵and R⁶ are independently H or C₁-C₆ alkyl or C₃-C₆ cycloalkyl.

In certain embodiments, compounds of formula (I) are provided wherein R⁵and R⁶ are independently H or C₃-C₆ cycloalkyl.

In certain embodiments, compounds of formula (I) are provided wherein R¹and R³ are H and R² is optionally substituted C₁-C₆ alkyl.

In certain embodiments, compounds of formula (I) are provided wherein R¹and R³ are H, R² is optionally substituted C₁-C₆ alkyl and R⁴ isoptionally substituted heteroaryl.

In certain embodiments, compounds of formula (I) are provided wherein R¹and R³ are H, R² is optionally substituted C₁-C₆ alkyl, R⁴ is optionallysubstituted heteroaryl, R⁵ and R⁶ are independently H or C₁-C₆ alkyl orare independently H and C₃-C₆ cycloalkyl, R⁷ is

wherein X is H, H and R⁹ is CONH₂.

In certain embodiments, compounds of formula (I) are provided wherein R⁴is an optionally substituted heteroaryl selected from the groupconsisting of quinoline, benzofuran, pyridine, benzothiophene,imidazopyridine and indazole.

Representative “pharmaceutically acceptable salts” of compounds offormula (I) include, for example, those of an acid or base. In oneembodiment, the pharmaceutically acceptable salt is selected from amongwater-soluble and water-insoluble salts. The pharmaceutically acceptablesalt can be of an acid selected from, e.g., among acetic, propionic,lactic, citric, tartaric, succinic, fumaric, maleic, malonic, mandelic,malic, phthalic, hydrochloric, hydrobromic, phosphoric, nitric,sulfuric, methanesulfonic, napthalenesulfonic, benzenesulfonic,toluenesulfonic, trifluoroacetic, and camphorsulfonic. Thepharmaceutically acceptable salt can also be of a base selected from,e.g., sodium, potassium, calcium, and ammonium. In some embodiments, acomposition, including a pharmaceutical composition, of the presentdisclosure can contain both a pharmaceutically acceptable salt and thefree base form of a compound of formula (I).

Compounds of formula (I) can also comprise a prodrug of formula (I).Prodrugs of compounds of formula (I) can be prepared using variousmethods known to those skilled in the art. See, e.g., Rautio, NatureReviews Drug Discovery, 7:255-270 (2008) and Ettcaner, J. Med. Chem.,47:2393-2404 (2004), which are hereby incorporated by reference. In thecase of drugs containing a hydroxy or carbonyl moiety, acetyl and otherester analogs are contemplated for use as prodrugs. See, e.g., Beaumont,Current Drug Metabolism, 4:461-485 (2003), which is hereby incorporatedby reference. In the case of drugs containing an amine moiety, prodrugscontaining amides and carbamates are contemplated. See, e.g., Simplicio,Molecules, 13:519-547 (2008), which is hereby incorporated by reference.As specific examples, (alkoxycarbonyloxy)alkyl carbamates,(acyloxy)alkyl carbamates, and (oxodioxolenyl)alkyl carbamates can beutilized as effective prodrug strategies for amines. See, e.g., Li,Bioorg. Med. Chem. Lett., 7:2909-2912 (1997); Alexander, J. Med. Chem.,34:78-81 (1991); Alexander, J. Med. Chem., 31:318-322 (1988); andAlexander, J. Med. Chem., 39:480-486 (1996), all of which areincorporated by reference herein.

Some compounds of formula (I) possess one or more chiral centers.Therefore, the disclosure of each compound includes its separateenantiomers as well as mixtures of the enantiomers (e.g., racemates).Where multiple chiral centers exist in compounds of formula (I), alsoprovided are each possible combination of chiral centers within acompound, as well as all possible enantiomeric and diastereomericmixtures thereof. All chiral, diastereomeric, and racemic forms of astructure are intended, unless the specific stereochemistry or isomericform is specifically indicated. Given the teachings herein, one ofordinary skill in the art could readily prepare optically active formsof compounds of formula (I), such as by resolution of racemic forms orby synthesis from optically active starting materials.

The following definitions are used in connection with the compoundsdescribed herein. In general, the number of carbon atoms present in agiven group is designated “C_(x) to C_(y)”, where x and y are the lowerand upper limits, respectively. The carbon number as used in thedefinitions herein refers to carbon backbone and carbon branching, butdoes not include carbon atoms of the substituents, such as alkoxysubstitutions and the like. Unless indicated otherwise, the nomenclatureof substituents that are not explicitly defined herein is determined bynaming from left to right the terminal portion of the functionalityfollowed by the adjacent functionality toward the point of attachment.As used herein, “optionally substituted” means that at least 1 hydrogenatom of the optionally substituted group has been replaced.

“Alkyl” refers to a hydrocarbon chain that can be straight or branched.In one embodiment, an alkyl contains 1 to 6 (inclusive) carbon atoms. Inanother embodiment, an alkyl contains 1 to 5 (inclusive) carbon atoms.In a further embodiment, an alkyl contains 1 to 4 (inclusive) carbonatoms. In yet another embodiment, an alkyl contains 1 to 3 (inclusive)carbon atoms. In still a further embodiment, an alkyl contains 1 or 2carbon atoms. Examples of alkyl groups that are hydrocarbon chainsinclude, for example, methyl, ethyl, propyl, butyl, pentyl, and hexyl,where all isomers of these examples are contemplated.

“Cycloalkyl” refers to carbocyclic rings that include, for example,cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like. In oneembodiment, the carbocyclic ring is 3- to 6-membered. In anotherembodiment, the carbocyclic ring is a 3- to 5-membered ring. In afurther embodiment, the carbocyclic ring is 4- to 6-membered. In still afurther embodiment, the carbocyclic ring is 3- or 4-membered, i.e.,cyclopropyl or cyclobutyl. Unless specifically noted, the alkyl groupsare unsubstituted, i.e., they contain carbon and hydrogen atoms only.However, when the alkyl group or carbocyclic ring is substituted, it isprefaced with the term “optionally substituted” or “substituted”. Theoptional substituents of the alkyl groups or carbocyclic rings caninclude, for example, halogen, CN, C₁ to C₆ alkyl, OH, C₁ to C₆ alkoxy,C₁ to C₆ alkoxy-C₁ to C₆ alkoxy, C₁ to C₆ alkoxy-C₁ to C₆ alkyl-C₁ to C₆alkoxy, heterocyclyloxy, aryl, heterocycle, heteroaryl, C(O)(C₁ to C₆alkyl), C(O)(heterocycle), C(O)NH₂, C(O)NH(C₁ to C₆ alkyl), C(O)N(C₁ toC₆ alkyl)(C₁ to C₆ alkyl), NHC(O)(C₁ to C₆ alkyl), NH₂, NH(aryl), N(C₁to C₆ alkyl)(C₁ to C₆ alkyl), and NHC(O)NH₂.

“Alkoxy” refers to

O(alkyl), where the alkyl is unsubstituted or substituted and is definedabove. In one embodiment, an alkoxy contains 1 to 6 (inclusive) carbonatoms or integers or ranges there between. In another embodiment, analkoxy contains 1 to 5 (inclusive) carbon atoms or ranges therebetween.In a further embodiment, an alkoxy contains 1 to 4 (inclusive) carbonatoms. In still a further embodiment, an alkoxy contains 1 to 3(inclusive) carbon atoms. In still a further embodiment, an alkoxycontains 1 or 2 carbon atoms. Examples of alkoxy include, for example,methoxy, ethoxy, propoxy, and butoxy. The alkyl radical of an alkoxygroup can be unsubstituted or substituted as defined above for “alkyl”.

“Aryl” refers to an aromatic hydrocarbon group containing carbon atoms.In one embodiment, the aryl contains 6 to 10 carbon atoms, i.e., 6-, 7-,8-, 9- or 10-membered. In another embodiment, aryl is an aromatic orpartly aromatic bicyclic group. In a further embodiment, the aryl is aphenyl group. In another embodiment, the aryl is naphthyl (such asα-naphthyl or β-naphthyl), 1,2,3,4-tetrahydronaphthyl, or indanyl. Anaryl group can be optionally substituted with one or more groupsincluding, without limitation, halogen, NO₂, C₁ to C₆ alkyl, OH, C₁ toC₆ alkoxy, C₁ to C₆ alkoxy-C₁ to C₆ alkoxy, C₁ to C₆ alkoxy-C₁ to C₆alkoxy-C₁ to C₆ alkoxy, heterocyclyloxy, C₁ to C₆ alkylthio, aryl,heterocycle, heteroaryl, C(O)(C₁ to C₆ alkyl), C(O)(heterocycle),C(O)O(C₁ to C₆ alkyl), C(O)NH₂, C(O)NH(C₁ to C₆ alkyl), C(O)N(C₁ to C₆alkyl)(C₁ to C₆ alkyl), SO₂(C₁ to C₆ alkyl), SO₂(C₂ to C₆ alkynyl),SO₂NH(C₁ to C₆ alkyl), SO₂(heterocycle), NHSO₂(C₁ to C₆ alkyl), N(C₁ toC₆ alkyl)SO₂(C₁ to C₆ alkyl), NH₂, NH(aryl) or NHC(O)NH₂.

“Halogen” refers to F, Cl, Br and I.

The term “heteroatom” refers to a sulfur, nitrogen, or oxygen atom. Itwill be apparent and understood by one of ordinary skill in the art thatthe chemical structures represented herein that contain heteroatoms may,in certain circumstances, have one or more bound hydrogens that are notshown. “Heteroaryl” refers to a monocyclic aromatic 5- or 6-memberedring containing one to four heteroatoms or heterogroups selected from—O—, —N—, —S—, —S(═O)—, —S(═O)₂, or —C(═O)—. In one embodiment, theheteroaryl contains 1 to 5 carbon atoms (inclusive) or integers orranges therebetween. In another embodiment, the heteroaryl contains 2 to5 carbon atoms (inclusive). In a further embodiment, the heteroarylcontains 3 to 5 carbon atoms (inclusive). In still a further embodiment,the heteroaryl contains 4 or 5 carbon atoms. “Heteroaryl” also refers tobicyclic aromatic ring systems wherein a heteroaryl group as justdescribed is fused to at least one other cyclic moiety. In oneembodiment, a phenyl radical is fused to a 5- or 6-membered monocyclicheteroaryl to form the bicyclic heteroaryl. In another embodiment, acyclic alkyl is fused to a monocyclic heteroaryl to form the bicyclicheteroaryl. In a further embodiment, the bicyclic heteroaryl is apyridine fused to a 5- or 6-membered monocyclic heteroaryl. In still afurther embodiment, the heteroaryl ring has 1 or 2 nitrogen atoms in thering. In still a further embodiment, the heteroaryl ring has 1 nitrogenatom and 1 oxygen atom. In still a further embodiment, the heteroarylring has 1 nitrogen atom and 1 sulfur atom. Examples of heteroarylgroups include, for example, furan, thiophene, indole, azaindole,oxazole, thiazole, isoxazole, isothiazole, imidazole, pyridine,pyridone, pyrazole, pyrimidine, pyrazine, pyridazine, pyrrole,oxadiazole such as 1,3,4-oxadiazole, triazole such as 1,2,4-triazole and1,2,3-triazole, tetrazole, benzoxazole, benzothiazole, benzofuran,benzisoxazole, benzimidazole, azabenzimidazole, indazole, quinazoline,quinoline, isoquinoline, benzothiophene and imidazopyridine. Aheteroaryl can be optionally substituted with one or more groupsincluding, without limitation, halogen, CN, NO₂, C₁ to C₆ alkyl, OH, C₁to C₆ alkoxy, C₃ to C₆ cycloalkyl, C₁ to C₆ alkoxy-C₁ to C₆ alkoxy, C₁to C₆ alkoxy-C₁ to C₆ alkoxy-C₁ to C₆ alkoxy, C₁ or C₆ alkyl containing1 to 3 fluorine atoms, C₁ to C₆ alkoxy containing 1 to 3 fluorine atoms,C₃ to C₆ cycloalkyl-C₁ to C₆ alkyl, C₁ to C₆ hydroxyalkyl,heterocyclyloxy, C₁ to C₆ alkylthio, aryl, heterocycle, heteroaryl,C(O)(C₁ to C₆ alkyl), C(O)(heterocycle), C(O)O(C₁ to C₆ alkyl), C(O)NH₂,C(O)NH(C₁ to C₆ alkyl), C(O)N(C₁ to C₆ alkyl)(C₁ to C₆ alkyl), SO₂(C₁ toC₆ alkyl), SO₂(C₂ to C₆ alkynyl), SO₂NH(C₁ to C₆ alkyl),SO₂(heterocycle), NHC(O)(C₁ to C₆ alkyl), NHSO₂(C₁ to C₆ alkyl), N(C₁ toC₆ alkyl)SO₂(C₁ to C₆ alkyl), NH₂, NH(aryl), N(C₁ to C₆ alkyl)(C₁ to C₆alkyl) and NHC(O)NH₂. In one embodiment, “heteroaryl” refers to5-membered heteroaryl containing one to four heteroatoms selected from—O—, —N—, or —S—, which is unsubstituted or substituted with one or morehalogen, C₁ to C₆ alkyl, C₃ to C₆ cycloalkyl, C₃ to C₆ cycloalkyl-C₁ toC₆ alkyl, C₁ to C₆ hydroxyalkyl, C₁ to C₆ alkoxy, C₁ to C₆ alkylcontaining 1 to 3 fluorine atoms, or CH₂CONH₂. Non-limiting examples of5-membered heteroaryl rings include, for example, oxadiazole, pyrazole,thiophene, isoxazole, imidazole, tetrazole, triazole, furan, pyrrole,thiazole, isothiazole, or thiadizole. Each of these 5-memberedheteroaryl can be substituted with one or more halogen, C₁ to C₆ alkyl,C₃ to C₆ cycloalkyl, C₃ to C₆ cycloalkyl-C₁ to C₆ alkyl, C₁ to C₆hydroxyalkyl, C₁ to C₆ alkoxy, C₁ to C₆ alkyl containing 1 to 3 fluorineatoms, or CH₂CONH₂.

“Heterocycle” refers to a monocyclic or bicyclic group having one tothree heteroatoms or heterogroups selected from —O—, —N—, —S—, —S(═O)—,—S(═O)₂, or —C(═O)—. A heterocycle can be saturated or partiallysaturated. In one embodiment, the heterocycle contains 3 to 7 carbonatoms (inclusive) or integers or ranges therebetween. In anotherembodiment, the heterocycle contains 4 to 7 carbon atoms (inclusive). Ina further embodiment, the heterocycle contains 4 to 6 carbon atoms(inclusive). In still a further embodiment, the heterocycle contains 5or 6 carbon atoms (inclusive). Examples of heterocycles include, forexample, to aziridine, oxirane, thiirane, morpholine, thiomorpholine,pyrroline, pyrrolidine, azepane, dihydrofuran, tetrahydrofuran,dihydrothiophene, tetrahydrothiophene, dithiolane, piperidine,1,2,3,6-tetrahydropyridine-1-yl, tetrahydropyran, pyran, thiane, thiine,piperazine, homopiperazine, oxazine, azecane, tetrahydroquinoline,perhydroisoquinoline, 5,6-dihydro-4H-1,3-oxazin-2-yl,2,5-diazabicyclo[2.2.1]heptane, 2,5-diazabicyclo[2.2.2]octane,3,6-diazabicyclo[3.1.1]heptane, 3,8-diazabicyclo[3.2.1]octane,6-oxa-3,8-diazabicyclo[3.2.1]octane,7-oxa-2,5-diazabicyclo[2.2.2]octane,2,7-dioxa-5-azabicyclo[2.2.2]octane,2-oxa-5-azabicyclo[2.2.1]heptane-5-yl, 2-oxa-5-azabicyclo[2.2.2]octane,3,6-dioxa-8-azabicyclo[3.2.1]octane, 3-oxa-6-azabicyclo[3.1.1]heptane,3-oxa-8-azabicyclo[3.2.1]octan-8-yl,5,7-dioxa-2-azabicyclo[2.2.2]octane,6,8-dioxa-3-azabicyclo[3.2.1]octane, 6-oxa-3-azabicyclo[3.1.1]heptane,8-oxa-3-azabicyclo[3.2.1]octan-3-yl,2,5-diazabicyclo[2.2.1]heptane-5-yl, 6-azabicyclo[3.2.1]oct-6-yl,8-azabicyclo[3.2.1]octan-8-yl, 3-oxa-7,9-diazabicyclo[3.3.1]nonan-9-yl,9-oxa-3-azabicyclo[3.3.1]nonan-3-yl,3-oxa-9-azabicyclo[3.3.1]nonan-9-yl,3,7-dioxa-9-azabicyclo[3.3.1]nonan-9-yl,3,4-dihydro-2H-1,4-benzoxazin-7-yl, thiazine, dithiane, and dioxane. Inanother embodiment, the heterocycle contains 1 or 2 nitrogen atoms. In afurther embodiment, the heterocycle contains 1 or 2 nitrogen atoms and 3to 6 carbon atoms. In still a further embodiment, the heterocyclecontains 1 or 2 nitrogen atoms, 3 to 6 carbon atoms, and 1 oxygen atom.In a still a further embodiment, the heterocycle is 5- to 8-membered. Instill a further embodiment, the heterocycle is 5-membered. In still afurther embodiment, the heterocycle is 6-membered. In still a furtherembodiment, the heterocycle is 8-membered. A heterocycle can beunsubstituted or substituted with one or more groups including, withoutlimitation, halogen, CN, NO₂, C₁ to C₆ alkyl, OH, C₁ to C₆ alkoxy, C₁ toC₆ alkoxy-C₁ to C₆ alkoxy, C₁ to C₆ alkoxy-C₁ to C₆ alkoxy-C₁ to C₆alkoxy, C₁ to C₆ hydroxyalkyl, C₃ to C₆ cycloalkyl, C₁ to C₆ alkylcontaining 1 to 3 fluorine atoms, C₃ to C₆ cycloalkyl-C₁ to C₆ alkyl,heterocyclyloxy, C₁ to C₆ alkylthio, aryl, heterocycle, heteroaryl,C(O)(C₁ to C₆ alkyl), C(O)(heterocycle), C(O)O(C₁ to C₆ alkyl), C(O)NH₂,C(O)NH(C₁ to C₆ alkyl), C(O)N(C₁ to C₆ alkyl)(C₁ to C₆ alkyl), SO₂(C₁ toC₆ alkyl), SO₂(C₂ to C₆ alkynyl), SO₂NH(C₁ to C₆ alkyl),SO₂(heterocycle), NHC(O)(C₁ to C₆ alkyl), NHSO₂(C₁ to C₆ alkyl), N(C₁ toC₆ alkyl)SO₂(C₁ to C₆ alkyl), NH₂, NH(aryl), N(C₁ to C₆ alkyl)(C₁ to C₆alkyl) and NHC(O)NH₂.

“Ester” refers to

C(O)O(alkyl), which is bound through the carbon atom. The alkyl group isdefined and unsubstituted or substituted as described above. Examples ofester include, without limitation, C(O)OCH₃, C(O)O(CH₂CH₃),C(O)O(CH₂CH₂CH₃), and C(O)(O)(CH₂CH₂CH₂CH₃).

It is to be noted that the term “a” or “an” refers to one or more. Assuch, the terms “a” (or “an”), “one or more,” and “at least one” areused interchangeably herein.

The words “comprise”, “comprises”, and “comprising” are to beinterpreted inclusively rather than exclusively. The words “consist”,“consisting”, and its variants, are to be interpreted exclusively,rather than inclusively.

As used herein, the term “about” means a variability of 10% from thereference given, unless otherwise specified.

As used herein, the terms “subject” and “patient” are usedinterchangeably and include any animal such as a mammal, e.g., a human,mouse, rat, guinea pig, dog, cat, horse, cow, pig, or non-human primate,such as a monkey, chimpanzee, baboon or gorilla.

As used herein, the terms “disease”, “disorder” and “condition” are usedinterchangeably, to indicate an abnormal state in a subject.

In one embodiment, the present disclosure provides a pharmaceuticalcomposition comprising a compound of formula (I). Pharmaceuticalcompositions of the present disclosure as described herein can comprisea compound of formula (I) optionally with one or more pharmaceuticallyacceptable excipients. Pharmaceutical compositions of the presentdisclosure can also comprise a compound of formula (I) and one or moretherapeutic agents, i.e., active ingredients, as described below. In afurther embodiment, Pharmaceutical compositions of the presentdisclosure comprise a compound of formula (I), one or morepharmaceutically acceptable excipients and one or more therapeuticagents.

The pharmaceutical compositions of the present disclosure can comprisean amount of a compound of formula (I) that is effective for regulatingthe P2X₃ or P2X_(2/3) pathways, for example to achieve a therapeuticeffect, such as the treatment of pain and/or respiratory dysfunctions ina subject. The amount of a compound of formula (I) administered to asubject (either alone or comprising a pharmaceutical composition) whichis effective for regulating the P2X₃ or P2X_(2/3) pathways to achieve atherapeutic effect is a “therapeutically effective amount.”

In some embodiments, the therapeutically effective amount of thecompounds of formula (I) will depend on factors such as the formulation,pharmacological potency of the drug, age, weight and sex of the patient,condition being treated, disease penetration and/or severity of thepatient's symptoms, specific compounds of formula (I), route ofdelivery, and response pattern of the patient. It is also contemplatedthat the treatment and dosage of the compounds of formula (I) can beadministered in a dosage form, and that one skilled in the art canadjust the dosage form accordingly to reflect the relative level ofactivity needed or desired to administer a therapeutically effectiveamount. The particular dosage to be employed (and also, for example, thenumber of times to be administered per day) to administer atherapeutically effective amount can be readily determined by anordinarily-skilled physician, and can be varied, for example, bytitration of the dosage to the particular circumstances in order toproduce a desired therapeutic effect. Further, the ordinarily-skilledphysician can readily calculate any changes needed or desired in theamounts of any one of the compounds of formula (I) to administer atherapeutically effective amount; for example, changes in components ordilutions of compounds or pharmaceutical compositions of the presentdisclosure. In one embodiment, the compounds or compositions can bediluted about 2-fold, 4-fold, or 8-fold prior to administration to asubject.

In one embodiment, the present disclosure provides a method of treatingpain in a subject, comprising administering to the subject atherapeutically effective amount of a compound of formula (I) (eitheralone or comprising a pharmaceutical composition). For treating painaccording to the present method, exemplary dosages to administer atherapeutically effective amount include the following: about 0.0001% toabout 25% w/v (i.e., weight of drug per mL of formulation); less thanabout 20% w/v, about 15% w/v, about 10% w/v, about 5% w/v, or about 1%w/v; about 0.0001% to about 10% w/v; about 0.005 to about 5% w/v. Incertain embodiments, a therapeutically effective amount of a compound offormula (I) is about 0.01 to about 5% w/v; about 0.01% w/v, about 0.05%w/v, about 0.1% w/v, about 0.2% w/v, about 0.3% w/v, about 0.4% w/v,about 0.5% w/v, about 0.6% w/v, about 0.7% w/v, about 0.8% w/v, about0.9% w/v, about 1% w/v, about 2% w/v, about 3% w/v, about 4% w/v, orabout 5% w/v. In one embodiment, the therapeutically effective amount ofa compound of formula (I) is about 0.2% w/v or about 0.5% w/v.

In some embodiments, the therapeutically effect amount of a compound offormula (I) administered to treat pain in a subject can be about 1 mg toabout 1000 mg per dose based on a 70 kg mammalian, for example human,subject. In another embodiment, the therapeutically effective amount isabout 2 mg to about 250 mg per dose or about 5 mg to about 100 mg perdose. In a further embodiment, the therapeutically effective amount isabout 25 mg to 50 mg, about 20 mg, about 15 mg, about 10 mg, about 5 mg,about 1 mg, about 0.1 mg, about 0.01 mg or about 0.001 mg per dose.

In one embodiment, the present disclosure provides a method of treatingrespiratory dysfunctions in a subject, comprising administering to thesubject a therapeutically effective amount of a compound of formula (I)(either alone or comprising a pharmaceutical composition). For treatingof respiratory dysfunctions according to the present method, exemplarydosages to administer a therapeutically effective amount of a compoundof formula (I) include the following: about 0.0001% to about 50% w/v(i.e., weight of drug per mL of formulation); about 0.5% to about 25%w/v; about 1% to about 20% w/v; or about 5% to 10% w/v. In a furtherembodiment, the therapeutically effective amount is about 0.01% w/v,about 0.05% w/v, about 0.1% w/v, about 0.2% w/v, about 0.3% w/v, about0.4% w/v, about 0.5% w/v, about 0.6% w/v, about 0.7% w/v, about 0.8%w/v, about 0.9% w/v, about 1% w/v, about 2% w/v, about 3% w/v, about 4%w/v, or about 5% w/v.

In some embodiments, the therapeutically effective amount of a compoundof formula (I) administered to treat respiratory dysfunction in asubject can be about 0.001 mg to about 1,000 mg per dose based on a 70kg mammalian, for example human. In one embodiment, a therapeuticallyeffective amount is about 1 mg to about 250 mg per dose or about 5 mg toabout 100 mg per dose. In some embodiments, a therapeutically effectiveamount of a compound of formula (I) comprises a dosage of about 0.1 mg,1 mg, about 5 mg, about 10 mg, about 15 mg, about 20 mg, about 25 mg,about 50 mg, about 100 mg, about 200 mg, about 300 mg, about 400 mg,about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg,about 1,000 mg per dose.

In methods of the present disclosure, a therapeutically effective amountof a compound of formula (I) can be administered (either alone orcomprising a pharmaceutical composition) to a subject on regularschedule, i.e., on a daily, weekly, monthly, or yearly basis or on anirregular schedule with varying administration days, weeks, months, etc.Alternatively, the therapeutically effective amount can be administeredin multiple doses, which can be divided equally or unequally. Each dosecan constitute a therapeutically effective amount, or the total amountdosed can constitute a therapeutically effective amount. For example inone embodiment, the therapeutically effective amount for the first dosecan be higher than the therapeutically effective amount for one or moreof the subsequent doses. In another embodiment, the therapeuticallyeffective amount for the first dose is lower than the therapeuticallyeffective amount for one or more of the subsequent doses. Equally orunequally divided doses can be administered over various time periodsincluding, for example, about every 2 hours, about every 6 hours, aboutevery 8 hours, about every 12 hours, about every 24 hours, about every36 hours, about every 48 hours, about every 72 hours, about every week,about every 2 weeks, about every 3 weeks, about every month, about every2 months, about every 3 months and about every 6 months. The number andfrequency of dosages corresponding to a completed course of therapy canbe determined according to the judgment of an ordinarily-skilledphysician. As mentioned above, the therapeutically effective amountsdescribed herein can refer to each dose of a compound of formula (I)given, or can refer to the total amounts administered for a given timeperiod; that is, if more than one compound of formula (I) isadministered to a subject, the therapeutically effective amounts cancorrespond to the total amount administered. The duration of treatmentcan last for days, weeks, months or years. In some embodiments,treatment lasts for two weeks. In some embodiments, treatment lasts onemonth. In some embodiments, treatment can proceed indefinitely.

In methods of the present disclosure, a therapeutically effective amountof a compound of formula (I) can be administered (either alone orcomprising a pharmaceutical composition) to a subject by any route,taking into consideration the specific condition being treated. Suitableroutes of administration include orally, by injection, inhalation(including orally, intranasally and intratracheally), ocularly,transdermally (e.g., via simple passive diffusion formulations or viafacilitated delivery using, for example, iontophoresis, microporationwith microneedles, radio-frequency ablation or the like),intravascularly, cutaneously, subcutaneously, intramuscularly,sublingually, intracranially, epidurally, rectally, intravesically, andvaginally, among others.

As discussed above, compounds of formula (I) can be administered to asubject alone according to the present methods, or these compounds canbe administered as a pharmaceutical composition. Pharmaceuticalcompositions of the present disclosure comprise a compound of formula(I) and one or more pharmaceutical excipients that are physiologicallycompatible with the subject to which they are administered. The presentpharmaceutical compositions can be in dry or liquid form. Thus in someembodiments, pharmaceutical excipient(s) for use in the presentpharmaceutical compositions can be solid or liquid and can incorporate,for example, both solid and liquid excipients/matrices. Pharmaceuticalcompositions comprising a compound of formula (I) can be formulated neator with one or more pharmaceutical excipients for administration to asubject, for example for administration to a human. The nature andamount of the pharmaceutical excipient(s) comprising the pharmaceuticalcompositions of the present disclosure can be determined by factors suchas the solubility and other chemical properties of the compounds offormula (I), chosen route of administration and standard pharmacologicalpractice. In some embodiments, pharmaceutical compositions of thepresent disclosure optionally contain one or more excipients and one ormore compounds of formula (I).

Examples of suitable excipients for use in pharmaceutical compositionsof the present disclosure include surfactants, adjuvants, antioxidants,binders, buffers, coatings, coloring agents, compression aids, diluents,disintegrants, emulsifiers (e g., polyoxyethylene fatty acid esters),emollients, encapsulating materials, fillers, flavoring agents,glidants, granulating agents, lubricants, metal chelators,osmo-regulators, pH adjustors (e.g., sodium hydroxide), preservatives,solubilizers, sorbents, stabilizing agents, sweeteners (such assaccharin), surfactants, suspending agents, syrups, thickening agents(e.g., carboxypolymethylene or hydroxypropylmethylcellulose),penetration enhancers (e.g., hydroxypolyethoxydodecane, DMSO, DMAC, DDM,etc.) or viscosity regulators (such as polymers to increase viscosity),for example as described in the “Handbook of Pharmaceutical Excipients”,5^(th) Edition, Eds.: Rowe, Sheskey, and Owen, APhA Publications(Washington, D.C.), Dec. 14, 2005, which is incorporated herein byreference.

In some embodiments, the compounds of formula (I) can be formulated intopharmaceutical compositions with one or more a solid excipients. In oneembodiments, the pharmaceutical compositions can be compacted into aunit dose form, i.e., tablet or caplet. In another embodiment, thepharmaceutical compositions can be added to unit dose form, i.e., acapsule. In a further embodiment, the pharmaceutical compositions can beformulated for administration as a powder. The solid excipientcomprising a pharmaceutical composition of the present disclosure canperform a variety of functions; i.e., can perform the functions of twoor more of the excipients described below. For example, a solidexcipient can act as a flavoring agent, lubricant, solubilizer,suspending agent, filler, glidant, compression aid, binder,disintegrant, or encapsulating material. In one embodiment, a solidexcipient acts as a lubricant, solubilizer, suspending agent, binder,disintegrant, or encapsulating material. Similarly, a variety ofsuitable solid (e.g., rigid or flexible) excipients and excipients arewell-known to those of skill in the art. Such suitable solid excipientscan also be designed so as to undergo a state transition when introducedinto the body, for example into a body cavity such as the bladder (e.g.,liquid to gel, liquid to solid, gel to solid), and such materials arewell-known to those skilled in the art. Such suitable solid excipientscan also comprise a membrane, for example comprising a thermoelasticpolymer, which defines a reservoir containing a solid or liquidpharmaceutical composition of the present disclosure. Other suitablesolid excipients can comprise a thermoelastic polymer matrix, in which apharmaceutical composition of the present disclosure is embedded. Thecompounds of formula (I) or a pharmaceutical composition of the presentdisclosure comprising a compounds of formula (I) can also beadministered to a subject together with other-membrane stabilizers(e.g., local anesthetics), for example to form eutectic mixtures.

Liquid pharmaceutical compositions of the present disclosure cancomprise sterile solutions or suspensions. When liquid excipients areutilized, they can be sterile liquids. Other suitable liquid excipientscan be utilized in preparing liquid pharmaceutical compositions of thepresent disclosure, for example formulated as solutions, suspensions,emulsions, syrups and elixirs. In one embodiment, a liquidpharmaceutical composition of the present disclosure comprises acompound of formula (I) dissolved a liquid excipient. In anotherembodiment, a liquid pharmaceutical composition of the presentdisclosure comprises a compound of formula (I) suspended in a liquidexcipient. A variety of suitable liquid excipients are well-known andcan be readily selected by one of skill in the art and can include,e.g., dimethylsulfoxide (DMSO), saline, buffered saline, cyclodextrin,hydroxypropylcyclodextrin (HPβCD), n-dodecyl-β-D-maltoside (DDM) andmixtures thereof.

In some embodiments, the present pharmaceutical compositions can besub-divided into unit dosage forms to contain appropriate quantities(e.g., therapeutically effective amounts) of a compound of formula (I).For example, the unit dosage can comprise packaged compositions, e.g.,packeted powders, vials, ampoules, prefilled syringes or sachetscontaining liquids.

In one embodiment, the pharmaceutical compositions of the presentdisclosure can be utilized as inhalants. For this route ofadministration, pharmaceutical compositions of the present disclosurecan be prepared as fluid unit doses comprising a compound of formula (I)and a vehicle for delivery, for example, by an atomizing spray pump, oras a dry powder comprising a compound of formula (I) for insufflation.

In another embodiment, pharmaceutical compositions of the presentdisclosure can be administered according to the present methods asaerosols; i.e., for oral or intranasal administration. For such routesof administration, the present pharmaceutical compositions can beformulated, for example, for use in a pressurized aerosol containertogether with a gaseous or liquefied propellant; e.g.,dichlorodifluoromethane, carbon dioxide, nitrogen, propane, and thelike, and can be delivered as a metered dose in one or more actuationsof a suitable delivery device. In certain embodiments, pharmaceuticalcompositions of the present disclosure formulated for inhalation cancomprise any well-known pharmaceutically acceptable medically inertexcipient, such as diluents, excipients, surfactants and flavorings.

In another embodiment, pharmaceutical compositions of the presentdisclosure can comprise an ingestible liquid, such as a syrup, elixir,solution, suspension, emulsion, microemulsion, nano-emulsion, colloid,liquid gel, or the like. In still another embodiment, the presentpharmaceutical compositions can be formulated for oral administration inan oral film, lozenge, drop, or chewable dosage form which can include,for example, a pill, gum and the like. Pharmaceutical compositions ofthe present disclosure formulated as oral dosage forms can be useful fortreating subjects that are young, elderly, or have swallowing issues, orfor subjects that are non-human animals.

In another embodiment, pharmaceutical compositions of the presentdisclosure can be formulated to achieve a modified-release of thecompound of formula (I). “Modified-release” as used herein refers todelivery of a therapeutically effective amount of a compound of formula(I) which is controlled, for example, to prevent immediate release or torelease the compound of formula (I) over an extended or sustainedperiod; for example over at least about 8 hours (e.g., extendeddelivery) to at least about 12 hours (e.g., sustained delivery).Pharmaceutical compositions of the present disclosure can also beformulated to permit immediate release of a therapeutically effectiveamount of a compound of formula (I) (e.g., where therapeutic levels areachieved in less than about 1 hour or in less than about 2 hours).

Compounds of formula (I) or pharmaceutical compositions of the presentdisclosure can also be administered to a subject according to thepresent methods by a modified release delivery device. Suitable modifiedrelease delivery devices include, for example, drug-eluting implants.Such implants can comprise a thermoelastic polymer matrix, such as asilicon or ethylene vinyl acetate matrix, wherein one or more compoundsof formula (I), optionally with one or more pharmaceutically acceptableexcipients, is embedded. Suitable implants are described, for example,in U.S. Pat. No. 7,736,665 and US Patent Publication No.US-2011/0280922, the disclosures of which are herein incorporated byreference.

Other suitable drug-eluting implants can comprise an “osmotic pump” orother mechanism by which a solution comprising one or more compounds offormula (I), or a pharmaceutical composition comprising a compound offormula (I), contained within the device is forced out, for examplethrough the implant walls or through one or more apertures, by osmoticpressure which builds within the device once it is implanted into asubject. Suitable osmotic pump drug eluting implants are described, forexample, in U.S. Pat. Nos. 5,035,891 and 6,464,688, the disclosures ofwhich are herein incorporated by reference.

Still other suitable drug-eluting implants can comprise a hydrogel suchas a polymethacrylate-based polymer (e.g., as described in U.S. Pat.Nos. 5,292,515 and 5,266,325, the disclosures of which are hereinincorporated by reference), or a thermoelastic polymer, such as apolyurethane (e.g., as described in U.S. Pat. Nos. 7,858,110 and7,842,303, the disclosures of which are herein incorporated byreference), which define a reservoir containing a solid or liquidcomposition comprising one or more compounds of formula (I) orpharmaceutical compositions of the present disclosure. Still otherdrug-eluting implants suitable for use in the present methods cancomprise a bio-degradable or bio-erodible polymer and at least one ormore compounds of formula (I) or pharmaceutical compositions of thepresent disclosure, for example as described in U.S. Pat. Nos. 4,906,474and 5,633,002, the disclosures of which are herein incorporated byreference.

Modified release of the compounds of formula (I) or a pharmaceuticalcomposition comprising a compounds of formula (I) can also be achievedby injecting the compounds or compositions into the bladder tissue(e.g., into the urothelium or muscularis propria) with a device that canbe employed via an endoscope inserted into the bladder orpercutaneously. For example, compounds of formula (I) or pharmaceuticalcompositions of the present disclosure can be injected into the bladdertissue via a needle, or a needleless device, for example as described inUS Patent Publication No. US-2011/0046600, the disclosure of which isincorporated by reference. A suitable needleless injection device foruse in the present methods includes the JetTouch™ platform (AmericanMedical Systems; Minnetonka, Minn.). The injected compounds of formula(I) or pharmaceutical compositions of the present disclosure can form adepot, and in certain embodiments, the injected compounds of formula (I)or pharmaceutical compositions of the present disclosure can beencapsulated in a bio-degradable or bio-erodible polymer, for example asdescribed in U.S. Pat. Nos. 5,480,656 and 6,036,976, the disclosures ofwhich are incorporated by reference.

Modified release of the compounds of formula (I) or pharmaceuticalcompositions of the present disclosure can also be achieved byinstilling the compounds or pharmaceutical compositions with at leastone material that solidifies or gels upon administration, for exampleonce instilled into the bladder or upon contact with the bladderurothelium, to coat at least a portion of the bladder wall. Thecompounds of formula (I) or pharmaceutical compositions of the presentdisclosure can then elute from the solidified or gelled material. See,e.g., U.S. Pat. Nos. 6,894,071; 5,575,815 and 6,039,967, the disclosuresof which are incorporated by reference, for examples of suitable gellingor solidifying materials.

In still a further embodiment, the compounds of formula (I) orpharmaceutical compositions of the present disclosure can beadministered transdermally, e.g., via the use of a drug-eluting patch.In one embodiment, the drug-eluting patch is an “iontophoretic”transdermal patch in drug delivery is effected using, e.g.,microprocessor-controlled, electrical current produced by, for example,an external source or an on-board battery. In a further embodiment, thedrug-eluting patch is a “microneedle” transdermal patch, which containsmicroneedles coated with or containing (e.g., in dissolvable ornon-dissolvable form) a compound of formula (I) or pharmaceuticalcomposition of the present disclosure. Suitable “microneedle”transdermal patches are described in, e.g., U.S. Pat. Nos. 7,798,987 and7,537,795, the disclosures of which are herein incorporated byreference. The microneedles can themselves be dissolvable ornon-dissolvable; see, for example, the “microneedle” technologydescribed in Sullivan, “Dissolving Polymer Microneedle Patches forInfluenza Vaccination”, Nature Medicine, 16:915-920 (Jul. 18, 2010online publication) and Lee, “Dissolving Microneedle Patch forTransdermal Delivery of Human Growth Hormone”, Small, 7(4):531-539 (Jan.4, 2011 online publication), which are herein incorporated by reference.

Other suitable transdermal delivery systems for use in the presentmethods include the radio-frequency ablation systems described inSintov, “Radiofrequency-Driven Skin Microchanneling as a New Way forElectrically Assisted Transdermal Delivery of Hydrophilic Drugs”,Controlled Release 89: 311-320 (2003), and U.S. Pat. No. 7,558,625, thedisclosures of which are herein incorporated by reference, and thetransdermal patches described in U.S. Pat. Nos. 5,411,738 and 5,827,528and Prausnitz and Langer, “Transdermal drug delivery”, NatureBiotechnology, 26(11):1261-1268, November 2006, which are hereinincorporated by reference.

In some embodiments, a transdermal delivery system for use according tomethods of the present disclosure can be selected which permits orassists a compound of formula (I) in passing through the dermal layerand to the targeted area, such as muscular tissues or a perineuralspace. Such systems can include formulation or use of a compound offormula (I) with skin penetration enhancers. Suitable skin penetrationenhancers include, for example, physical enhancers (ultrasound,iontophoresis, electroporation, magnetophoresis, microneedle), vesicles,particulate systems (liposome, niosome, transfersome, microemulsion,solid lipid nanoparticle), and chemical enhancers (sulfoxides, azones,glycols, alkanols, terpenes, etc.). Other suitable chemical enhancersinclude, e.g., propylene glycol, polyethylene glycol, isopropanol,ethanol, oleic acid, N-methylpyrrolidone, which can increase thepermeability of the skin to the compounds, and permit the compounds topenetrate through the skin to deeper tissues. Suitable examples ofchemical skin penetration enhancers for use in the present methods arealso described in, for example, Sagie & Kohane, “ProlongedSensory-Selective Nerve Blockade”, PNAS, 2010(8): 3740-3745, 2010, whichis herein incorporated by reference.

In one embodiment, a transdermal patch for use according to the presentmethods is applied via a suitable adhesive on the skin, where it remainsin place for at least one hour. In a further embodiment, the patchremains in place for about 1 hour and is replaced weekly, for a total ofabout 2 or about 3 hours wear time. In another embodiment, the patchremains in place for about 2 hours, about 3 hours, about 4 hours, about6 hours, about 12 hours or about 24 hours. In yet another embodiment,the patch remains in place for longer or shorter periods of time.

In certain embodiments of treatment methods of the present disclosure, acompound of formula (I) can be administered in combination with one ormore other medications or therapeutic agents. In one embodiment, acompounds of formula (I) can be combined with other medications ortherapeutic agents in a single pharmaceutical composition. In otherembodiments, a compound of formula (I) can be administered in one ormore separate formulations from other compounds of formula (I), or othermedications or therapeutic agents as described below. For treating painand respiratory dysfunction, these other medications or therapeuticagents for administration in combination with the compounds of formula(I) can include, for example, TRPV1 and TRPA receptor activators andinhibitors, inhibitors of voltage-gated ion channels, non-steroidalanti-inflammatory drugs (NSAIDs), steroids, inhibitors of SpleenTyrosine Kinase and the JAK-STAT pathway, cytokine inhibitors ormodulators, opioids, tricyclic antidepressants, amine transporterinhibitors, and anticonvulsants (such as gabapentinoids). Othermedications or therapeutic agents that can be used in combination withcompounds of formula (I) for treating respiratory conditions includeanticholinergic agents and beta-receptor agonists.

In one embodiment, methods of the present disclosure compriseadministering to a subject a compound of formula (I), or pharmaceuticalcompositions comprising a compound of formula (I), with a with a TRPV1receptor activator for regulating activity of either or both of the P2X₃or P2X_(2/3) pathways. Regulation of either or both of the P2X₃ orP2X_(2/3) pathways can result in the treatment of pain or respiratorydysfunction in a subject. The term “TRPV1 receptor activator” as usedherein refers to any agent or stimulus that activates TRPV1 receptors onnociceptors or puriceptors, and allows for entry of at least oneinhibitor of voltage-gated ion (e.g., sodium or calcium) channels. Inone embodiment, the TRPV1 receptor activator includes, for example,capsaicin, dihydrocapsaicin and nordihydrocapsaicin, lidocaine,articaine, procaine, tetracaine, mepivicaine, bupivicaine, eugenol,camphor, clotrimazole, arvanil (N-arachidonoylvanillamine), anandamide,2-aminoethoxydiphenyl borate (2APB), AM404, resiniferatoxin, phorbol12-phenylacetate 13-acetate 20-homovanillate (PPAHV), olvanil (NE19550), OLDA (N-oleoyldopamine), N-arachidonyldopamine (NADA),6′-iodoresiniferatoxin (6′-IRTX), Cl 8 N-acylethanolamines, lipoxygenasederivatives (such as 12-hydroperoxyeicosatetraenoic acid), inhibitorcysteine knot (ICK) peptides (vanillotoxins), MSKl 95(N-[2-(3,4-dimethylbenzyl)-3-(pivaloyloxy)propyl]-2-[4-(2-aminoethoxy)-3-methoxyphenyl]acetamide),JYL79(N-[2-(3,4-dimethylbenzyl)-3-(pivaloyloxy)propyl]-N′-(4-hydroxy-3-methoxybenzyl)thiourea),hydroxy-α-sanshool, 2-aminoethoxydiphenyl borate, 10-shogaol,oleylgingerol, oleylshogaol, SU200(N-(4-tert-butylbenzyl)-N′-(4-hydroxy-3-methoxybenzyl)thiourea)nonivamide, and fatty acyl amides of tetrahydroisoquinolines. In anotherembodiment, the TRPV1 receptor activator is lidocaine, aprindine,benzocaine, butacaine, cocaine, dibucaine, encainide, mexiletine,oxetacaine (oxethazaine), prilocaine, proparacaine, procainamide,n-acetylprocainamide, chloroprocaine (nesacaine, nescaine), dyclonine,etidocaine, levobupivacaine, ropivacaine, cyclomethycaine, dimethocaine(larocaine), propoxycaine, trimecaine, and sympocaine. In a furtherembodiment, the TRPV1 receptor activator is lidocaine. In anotherembodiment, the TRPV1 activator can be a detergent or a surfactant,examples of which can be found in commonly-used hygiene products such assoaps and shampoos (e.g., sodium lauryl sulfate), for example asdescribed in Lilja, “Surfactant-Induced TRPV1 activity—A Novel Mechanismfor Eye Irritation?” Technological Sciences, 99(1):174-180, 2007, whichis incorporated herein by reference. In another embodiment, the TRPV1receptor activator is not a chemical agent, but is heat or inflammation,both of which are known to activate TRPV1 receptors.

In one embodiment, the amount of the TRPV1 receptor activator used inthe present methods is about 0.0001% to about 10% w/v. One of skill inthe art would readily understand that the recited TRPV1 receptoractivator amount can be based, where appropriate, on the free base ofthe TRPV1 receptor activator. In some embodiments, the TRPV1 receptoractivator amount is less than about 10% w/v, about 9% w/v, about 8% w/v,about 7% w/v, about 6% w/v, about 5% w/v, about 4% w/v, about 3% w/v,about 2% w/v, or about 1% w/v. In another embodiment, thetherapeutically effective amount is about 0.1% to about 5% w/v, forexample about 0.5 to about 3% w/v; about 0.5 to about 2% w/v; or about2% w/v. In another embodiment, the TRPV1 receptor activator amount isabout 1% w/v or about 0.5% w/v.

In some embodiments, the amount of the TRPV1 receptor activator can beabout 0.001 mg to about 100 mg per dose based on a 70 kg mammaliansubject, for example about 0.1 mg to about 25 mg per dose; or about 1 mgto about 5 mg per dose. In other embodiments, the amount of the TRPV1receptor activator can be about 0.1 mg, about 0.5 mg, about 1 mg, about2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, orabout 8 mg per dose.

In one embodiment, a pharmaceutical composition of the presentdisclosure comprises a compound of formula (I) and lidocaine; forexample about 0.01% to about 1% w/v of a compound of formula (I) andabout 0.1% to about 5% w/v of lidocaine; about 0.1% to about 0.7% w/v ofa compound of formula (I) and about 1% to about 3% w/v of lidocaine;about 0.2% to about 0.5% w/v of a compound of formula (I) and about 1%to about 3% w/v of lidocaine; about 0.2% to about 0.5% w/v of a compoundof formula (I) and about 2% w/v of lidocaine; about 0.2% w/v of acompound of formula (I) and about 2% w/v of lidocaine; or about 0.5% w/vof a compound of formula (I) and about 2% w/v of lidocaine. As discussedabove, these compositions can be further diluted. In one embodiment,these pharmaceutical compositions can be diluted 2-fold. In anotherembodiment, these pharmaceutical compositions can be diluted 4-fold.

Also contemplated for use in the present methods are pharmaceuticalcompositions comprising a compound of formula (I) and inhibitors ofvoltage-gated ion channels. In one embodiment, the voltage-gated ionchannels are sodium or calcium ion channels. Suitable voltage-gatedsodium channel inhibitors include, for example, QX-314,N-methyl-procaine (QX-222), N-octyl-guanidine, 9-aminoacridine, andpancuronium. Suitable voltage-gated calcium channel inhibitors include,for example, D-890 (quaternary methoxyverapamil) and CERM 1 1888(quaternary bepridil). Other suitable voltage-gated ion channelinhibitors include, for example, riluzole, mexilitine, phenytoin,carbamazepine, procaine, tocainide, prilocaine, diisopyramide,bencyclane, quinidine, bretylium, lifarizine, lamotrigine, flunarizine,articaine, bupivicaine, mepivicaine, fluspirilene, orphenadrine,phenbenzamine, bepridil, pimozide, penfluridol, fluspirilene,propiverine, disopyramide, methadone, tolterodine, tridihexethyl salts,tripelennamine, mepyramine, brompheniramine, chlorpheniramine,dexchlorpheniramine, carbinoxamine, levomethadyl acetate, gallopamil,verapamil, devapamil, tiapamil, emopamil, dyclonine, pramoxine,lamotrigine, mibefradil, gabapentin, amiloride, diltiazem, nifedipine,nimodipine, nitrendipine, cocaine, mexiletine, propafenone, quinidine,oxethazaine, articaine, riluzole, bencyclane, lifarizine, andstrychnine.

Pharmaceutical compositions of the present disclosure can also comprisea compound of formula (I) and a membrane permeable inhibitor ofvoltage-gated ion channels. Suitable membrane permeable inhibitor ofvoltage-gated ion channels include, for example, cocaine, carbamazepine,disopyramide, lamotrigine, procainamide, phenytoin, oxcarbazepine,topiramate, zonisamide, tetracaine, ethyl aminobenzoate, prilocaine,disopyramide phosphate, flecainide acetate, mexiletine, propafenone,quinidine gluconate, quinidine polygalacturonate, chloroprocaine,dibucaine, dyclonine, mepivacaine, pramoxine, procaine, tetracaine,oxethazaine, propitocaine, levobupivacaine, bupivacaine, lidocaine,moricizine, tocainide, proparacaine, ropivacaine, quinidine sulfate,encainide, ropivacaine, etidocaine, moricizine, quinidine, encainide,flecainide, tocainide, fosphenytoin, chloroprocaine, dyclonine,L-(−)-1-butyl-2′,6′-pipecoloxylidide and pramoxine.

Pharmaceutical compositions of the present disclosure can also comprisea compound of formula (I) and a vasoconstrictor, e.g., epinephrine orvasopressin or (for example when formulated as injectable solutions) cancomprise glucose or dextrose and a compound of formula (I), and can beadministered as an infusion or as a regional analgesic or anti-pruritic.

In some embodiments, the present disclosure also provides regimens, kitsor packages comprising a compound of formula (I) or pharmaceuticalformulations comprising the compounds of formula (I), or one or moredosage forms thereof, optionally together with instructions for storageand/or use.

The kits of the present disclosure can further comprise packaging or acontainer holding the present compounds or pharmaceutical compositionsformulated for a given route of administration. The kit can optionallycomprise instructions on dosing and/or an insert regarding the compoundsof formula (I). The optional instructions can, for example, containinformation regarding assays for monitoring local or circulating levelsof compounds of formula (I) or their metabolites, and the kit canfurther comprise materials for performing such assays including, e.g.,reagents, well plates, containers, markers or labels, and the like.

Kits of the present disclosure can be readily packaged in a mannersuitable for treatment of an indication. For example, the kit cancomprise a patch, spray pump, nasal spray, inhaler (including aerosol,metered dose, and dry powder inhalers, nebulizer, or other suitabledevice, and optionally instructions for their use. Other suitablecomponents to include in kits of the present disclosure will be readilyapparent to one of skill in the art, taking into consideration theindication and the delivery route, and can comprise, for example,lubricants, antiseptic solutions and local anesthetic agents tofacilitate the placement and use of the delivery device.

As discussed above, the compounds of formula (I) or pharmaceuticalcompositions of the present disclosure can be administered to a subjectin single dose or in multiple doses, for example for continuous orperiodic discontinuous administration. For continuous administration, apackage or kit of the present disclosure can comprise a compound offormula (I) or a pharmaceutical composition comprising a compound offormula (I) in one or more dosage units; e.g., solution, lotion, tablet,pill, drug-eluting unit/patch or other unit dosage form described aboveor which can be utilized in drug delivery, and optionally instructionsfor administering the doses (for example less-than-daily, daily, weekly,or monthly) for a predetermined length of time or as prescribed. Whenthe compounds or pharmaceutical compositions of the present disclosureare to be delivered periodically in a discontinuous fashion, a packageor kit can comprise placebos during periods when the compounds offormula (I) or pharmaceutical compositions are not delivered. Whentreatment methods of the present disclosure comprise varyingconcentrations of a composition, of the components of the composition,or the relative ratios of the compounds of formula (I) or agents orexcipients comprising a pharmaceutical a composition over time, apackage or kit can contain a sequence of dosage units which provide thevariability.

A number of packages for dispensing pharmaceutical agents for periodicoral use suitable for use in kits of the present disclosure arewell-known in the art. In one embodiment, the package can compriseindicators for each period. In another embodiment, the package comprisesa foil or blister package, labeled ampoule, vial or bottle.

In some embodiments, the kit of the present disclosure can itself beused to effect administration of a compound of formula (I) or apharmaceutical composition comprising a compound of formula (I), and cancomprise a drug delivery device such as an inhaler, syringe, pipette,eye dropper, catheter, cytoscope, trocar, cannula, pressure ejectiondevice, or other such apparatus, for example from which the presentcompounds or pharmaceutical compositions can be administered to asubject, (e.g., by application to an affected area of the subject'sbody).

A compound of formula (I), or a pharmaceutical composition comprising acompound of formula (I), comprising a kit of the present disclosure canbe provided in dried or lyophilized forms, for reconstitution by theaddition of a suitable solvent, which solvent can be provided with thekit. In some embodiments, the suitable solvent also can be provided inanother package.

In some embodiments, the kits of the present disclosure can comprise ameans (e.g., vials or other suitable packaging means) for containingcompounds of formula (I), or pharmaceutical compositions comprising acompound of formula (I), in close confinement for commercial sale suchas, e.g., injection or blow-molded plastic containers.

The compounds of formula (I) (either alone or comprising pharmaceuticalformulations) are useful in regulating the the P2X₃ and/or P2X_(2/3)pathways, which regulation can result in treating conditions which areassociated with the P2X₃ and/or P2X_(2/3) pathways, such as pain orrespiratory dysfunction. The term “regulation,” “modulation” orvariations thereof, as used herein, are used interchangeably and referto the ability of a compound of formula (I) to inhibit or reduce theactivity of one or more components of a biological pathway. In oneembodiment, “regulation” refers to inhibition of P2X₃ activity. Inanother embodiment, “regulation” refers to inhibition of P2X_(2/3)activity. In a further embodiment, “regulation” refers to dualinhibition of P2X₃ and P2X_(2/3) activity.

Accordingly, in one embodiment, the present disclosure provides a methodof treating pain in a subject, comprising administering to the subject atherapeutically effective amount of a compound of formula (I) (eitheralone or comprising a pharmaceutical formulation). Any type of pain canbe treated by the methods of the present disclosure. The term “pain” asused herein thus includes all types of pain, for example acute orchronic. In another embodiment, the pain can be somatic, central,visceral, idiopathic, dysfunctional, nociceptive, neuropathic,inflammatory, and/or procedural pain.

“Somatic pain” includes pain from bone, joint, muscle, skin, orconnective tissue.

“Central pain” includes pain arising as a consequence of brain trauma,stroke, or spinal cord injury.

“Visceral pain” includes pain from visceral organs, such as therespiratory or gastrointestinal tract and pancreas, the urinary tractand reproductive organs. In one embodiment, visceral pain results fromtumor involvement of the organ capsule. In another embodiment, visceralpain results from obstruction of hollow viscus. In a further embodiment,visceral pain results from inflammation as in cystitis or refluxesophagitis.

“Idiopathic pain” refers to pain which has no underlying cause or refersto pain caused by condition which remains undiagnosed.

“Dysfunctional pain” refers to pain which occurs in the absence of anoxious stimulus, tissue damage or a lesion to the nervous system. Inone embodiment, dysfunctional pain results from rheumatologic conditionssuch as arthritis and fibromyalgia, tension type headache, irritablebowel disorders and erythermalgia.

“Nociceptive pain” includes pain caused by noxious stimuli that threatento or actually injure body tissues. In one embodiment, nociceptive painresults from a cut, bruise, bone fracture, crush injury, burn, trauma,surgery, labor, sprain, bump, injection, dental procedure, skin biopsy,or obstruction. In another embodiment, nociceptive pain is located inthe skin, musculoskeletal system, or internal organs.

“Neuropathic pain” is pain due to abnormal processing of sensory inputby the peripheral or central nervous system consequent on a lesion tothese systems. In one embodiment, neuropathic pain is chronic andnon-malignant. In one embodiment, neuropathic pain is due to trauma,surgery, herniation of an intervertebral disk, spinal cord injury,diabetes, infection with herpes zoster (shingles), HIV/AIDS, late-stagecancer, amputation (such as mastectomy), carpal tunnel syndrome, chronicalcohol use, exposure to radiation, and as an unintended side-effect ofneurotoxic treatment agents, such as certain anti-HIV andchemotherapeutic drugs. In another embodiment, neuropathic pain is canbe described as “burning,” “electric,” “tingling,” or “shooting”.

“Inflammatory pain” includes pain resulting from inflammation caused byany number of factors. In one embodiment, inflammatory pain occurs dueto tissue damage or inflammation. In another embodiment, inflammatorypain is due to injury (including joints, muscle, and tendons injuries),surgical procedures, infection, and/or arthritis.

“Procedural pain” refers to pain arising from a medical procedure. Themedical procedure can include any type of medical, dental or surgicalprocedure. In one embodiment, the procedural pain is postoperative. Inanother embodiment, the pain is associated with an injection, drainingan abscess, surgery, dermatological, dental procedure, ophthalmicprocedure, arthroscopy and use of other medical instrumentation, and/orcosmetic surgery.

For example, the pain can be from a migraine, back pain, neck pain,gynecological pain, pre-labor or labor pain, orthopedic pain,post-stroke pain, post-surgical or procedural pain, post herpeticneuralgia, sickle cell crises, interstitial cystitis, urological pain(such as urethritis), dental pain, headache, pain from a wound or from amedical procedure such as surgery (such as bunionectomy or hip, knee orother joint replacement), suturing, setting a fracture, biopsy, and thelike. Pain can also occur in patients with cancer, which can be due tomultiple causes, such as inflammation, nerve compression, and mechanicalforces resulting from tissue distension as a consequence of invasion bya tumor and tumor metastasis into bone or other tissues. In oneembodiment, the cancer is bone cancer.

In one embodiment, the pain is neuropathic pain, such as post-herpeticneuralgia. In another embodiment, the pain is inflammatory pain. In afurther embodiment, the pain is nociceptive pain. In still anotherembodiment, the pain is procedural pain. In yet a further embodiment,the pain is caused by esophageal cancer, colitis, cystitis, irritablebowel syndrome, colitis or idiopathic neuropathy. In still anotherembodiment, the pain is caused by airway, bladder or visceral organdysfunction.

The term “treat”, “treating”, or any variation thereof is meant toinclude any therapy utilized to stabilize, lessen or ameliorate a healthproblem or condition in a patient or subject. In one embodiment, thehealth problem or condition can be eliminated permanently or for ashorter period of time. In another embodiment, the severity of thehealth problem or condition, or of one or more symptoms characteristicof the health problem or condition, can be delayed, kept from worsening,prevented from occurring, or lessened permanently or for a shorterperiod of time. The effectiveness of a treatment of pain can bedetermined using any standard pain index, such as those describedherein, or can be determined based on the patient's subjective pain. Apatient can be considered “treated” if there is a reported reduction inpain or a reduced reaction to stimuli that should cause pain.

Thus for the treatment of pain according to the present methods, asubject is treated for pain, for example, if there is a measurablechange in a relevant biological or chemical marker (including bymeasurement of a change in degree of binding affinity, etc. of such amarker) that is consistent with a reduction in pain, or if the subjectexperiences or is observed to experience a reduction in pain frequency,duration or intensity, or an improved quality of life apparently due toreduction in pain. For the treatment of respiratory dysfunctionaccording to the present methods, a subject is treated for respiratorydysfunction if there is a measurable change in a relevant biological orchemical marker (including by measurement of a change in degree ofbinding affinity, etc. of such a marker) that is consistent with areduction in one or more symptoms or a lessening of the severity of arespiratory condition, for example if the subject experiences or isobserved to experience less frequent or less intense cough, laboredbreathing, etc.

In order to measure the usefulness or effectiveness of any the treatmentmethods of the present disclosure, any suitable measurement index can beused. Indices that are suitable for the measurement of pain associatedwith musculoskeletal, immunoinflammatory and neuropathic disordersinclude a visual analog scale (VAS), a Likert scale, categorical painscales, subjective patient descriptors, the Lequesne index, the WOMACindex, and the AUSCAN index, each of which is well known in the art.Such indices can be used to measure pain, function, stiffness, or othervariables.

Indices that are useful of the measurement of pain associated withinterstitial cystitis include the interstitial cystitis symptom index(ICSI), the interstitial cystitis problem index (ICPI), thepain-urgency-frequency score (PUF), the Wisconsin Symptom Instrument(UWI) and a visual analog scale (VAS) such as the Likert scale and othercategorical pain scales.

A visual analog scale (VAS) provides a measure of a one-dimensionalquantity. A VAS generally utilizes a representation of distance, such asa picture of a line with hash marks drawn at regular distance intervals,e.g., ten 1-cm intervals. For example, a subject can be asked to rank asensation of pain by choosing the spot on the line that best correspondsto the sensation of pain, where one end of the line corresponds to “nopain” (score of 0 cm) and the other end of the line corresponds to“unbearable pain”. This procedure provides a simple and rapid approachto obtaining quantitative information about how the patient isexperiencing pain. VAS scales and their use are described, e.g., in U.S.Pat. Nos. 6,709,406 and 6,432,937, the relevant disclosures of which areherein incorporated by reference.

A Likert scale similarly provides a measure of a one-dimensionalquantity. Generally, a Likert scale has discrete integer values rangingfrom a low value, e.g., 0, meaning no pain, to a high value, e.g., 7,meaning extreme pain. A patient experiencing pain is asked to choose anumber between the low value and the high value to represent the degreeof pain experienced. Likert scales and their use are described, e.g., inU.S. Pat. Nos. 6,623,040 and 6,766,319, the relevant disclosures ofwhich are herein incorporated by reference.

The Lequesne index and the Western Ontario and McMaster Universities(WOMAC) osteoarthritis (OA) index assess pain, function, and stiffnessin the knee and hip of OA patients using self-administeredquestionnaires. Both knee and hip are encompassed by the WOMAC, whereasthere is one Lequesne questionnaire for the knee and a separate one forthe hip. These questionnaires are useful because they contain moreinformation content in comparison with VAS or Likert scale. Both theWOMAC index and the Lequesne index questionnaires have been extensivelyvalidated in OA, including in surgical settings, e.g., knee and hiparthroplasty, and their metric characteristics do not differsignificantly.

The AUSCAN (Australian-Canadian hand arthritis) index employs a valid,reliable, and responsive patient self-reported questionnaire. In oneinstance, this questionnaire contains 15 questions within threedimensions (Pain, 5 questions; Stiffness, 1 question; and Physicalfunction, 9 questions). An AUSCAN index can utilize, e.g., a Likert or aVAS scale.

The O'Leary-Sant score and IC Problem Index are self-administeredindices for measuring lower urinary tract symptoms.

The Pain-Urgency-Frequency symptom scale is balanced assessment ofurinary dysfunction, pelvic pain and symptoms associated with sexualintercourse and frequently used in conjunction with intravesicalpotassium chloride administration.

The UWI utilizes seven IC-related questions about frequency, urgency,noctuira and pain.

In addition to the indices discussed above, other suitable indices thatare useful for the measurement of pain include the Pain Descriptor Scale(PDS), the Verbal Descriptor Scales (VDS), the Numeric Pain IntensityScale (NPIS), the Neuropathic Pain Scale (NPS), the Neuropathic PainSymptom Inventory (NPSI), the Present Pain Inventory (PPI), theGeriatric Pain Measure (GPM), the McGill Pain Questionnaire (MPQ), meanpain intensity (Descriptor Differential Scale), numeric pain scale (NPS)global evaluation score (GES) the Short-Form McGill Pain Questionnaire,the Minnesota Multiphasic Personality Inventory, the Pain Profile andMultidimensional Pain Inventory, the Child Heath Questionnaire, and theChild Assessment Questionnaire.

In another embodiment, the present disclosure provides a method oftreating respiratory dysfunction in a subject, comprising administeringto the subject a therapeutically effective amount of a compound offormula (I) (either alone or comprising a pharmaceutical composition).The term “respiratory dysfunction” as used herein includes, for example,any disruption of normal respiratory function in a subject, such asbronchial hyperactivity, bronchoconstriction, bronchospasm,hypersecretion, cough, cough hypersensitivity syndrome, wheezing,dyspnea, breathless, and chest tightness, for example due to arespiratory disease or disorder. The term “respiratory disease ordisorder” as used herein includes, for example, idiopathic pulmonaryfibrosis (IPF), chronic obstructive pulmonary disease (COPD), asthma,upper respiratory infection, interstitial lung disease (ILD), post-nasaldrip, and bronchitis. The respiratory dysfunction can also be associatedwith gastroesophageal reflux disease (GERD), or can be an iatrogeniccough, including cough associated with treatment with an ACE(Angiotensin Converting Enzyme) inhibitor, or can be “smoker's cough”;that is, cough associated with smoking or exposure to smoke, dust, ashor the like. The term “cough” as used herein thus refers to any coughincluding, for example, sub-acute cough, chronic cough,treatment-resistant cough, idiopathic chronic cough, cough associatedwith upper respiratory infection, post-viral cough, iatrogenic cough,smoker's cough, cough associated with bronchitis, post-nasal drip or anyother irritation of the bronchi or esophagus, the urge to coughassociated with any respiratory disease, cough-variant asthma,interstitial lung disease, and whooping cough.

The terms “acute cough” refers to a cough lasting up to two weeks induration. For instance, acute cough can be the result of an acutedisease, such as a cold or flu. An acute cough will disappear when theunderlying cause (e.g., cold or flu) is eliminated.

The terms “sub-acute cough” refers to a cough lasting between two andeight weeks. In some cases, a sub-acute cough follows a period in whicha subject is infected with a disease (e.g., cold or flu). A sub-acutecough is one that often remains after the underlying cause has beenremoved. For instance, a sub-acute cough is found post-infection (e.g.,post-viral infection).

The terms “chronic cough” refers to a persistent or refractory coughlasting longer than eight weeks that does not have an obvious underlyingcause and can not be associated with other respiratory diseases, such asasthma or COPD. Chronic cough also generally has no hallmarks to defineand diagnose it, in contrast to other respiratory diseases (e.g., COPD),and a subject suffering from chronic cough can be apparently normal inmost other aspects. Chronic cough is characterized by frequent coughing(e.g., at least 5-10 coughs per hour during daytime) and bothersomecoughing during sleep. Chronic cough can last for a period of years,including over a decade.

Various tools have been developed to assess cough in clinical practiceand in clinical studies. For example, the visual analog scale (VAS) asdescribed above for the assessment of pain, is also widely used for theassessment of cough severity. The Leicester cough questionnaire (LCQ)and the cough-specific quality of life questionnaire (CQLQ) are alsoused to assess the impact of chronic cough. Ambulatory devicesconsisting of a microphone and recording device, such as the Leicestercough monitor (LCM) and the VitaloJak, are effective tools to measurecough frequency, particularly in clinical studies. Use of these toolscan provides evidence to measure a reduction in cough frequency and/orseverity for a patient, following treatment of a subject according tothe present methods.

In one embodiment, the present disclosure provides a method of treatinga respiratory condition in a subject comprising administering atherapeutically effective amount of a compound of formula (I) (eitheralone or comprising a pharmaceutical composition), wherein therespiratory condition is selected from the group consisting of acutecough, chronic cough, and cough associated with idiopathic pulmonaryfibrosis (IPF). Treatment of the respiratory condition can be measured,for example, by one or more techniques selected from the groupconsisting of the visual analog scale (VAS) for cough severity, theLeicester cough questionnaire (LCQ), the cough-specific quality of lifequestionnaire (CQLQ) and the Leicester cough monitor (LCM).

The following examples are illustrative only and are not intended tolimit the present disclosure.

EXAMPLES

Unless otherwise stated, all the raw materials are purchased fromcommercially available common suppliers. ¹H-NMR spectra were recordedusing tetramethylsilane (TMS) as the internal reference for CDCl₃dissolved compounds. For DMSO-d₆, MeOD and D₂O dissolved compounds theinstrument was calibrated at δ 2.5, 3.3 and 4.82 ppm respectively. Thechemical shift values are quoted in 6 (parts per million).

For LCMS analysis LCMS/MS API 2000 (Applied Biosystem) instrument wasused. The columns included:

Column V: Zorbax® C18 column, 4.6×50 mm, 5μ

Column W: Zorbax® Extend C18 column, 4.6×50 mm, 5μ

Column X: Gemini® NX C18 column, 4.6×50 mm, 5μ

Column Y: Xbridge® C18 column, 4.6×50 mm, 5μ

Column Z: Reprosil® column, 4.6×50 mm, 5μ

The eluent (solvent) typically included (acidic or basic buffer asaqueous phase):

A channel: (i) 0.05% formic acid in water; (ii) 10 mM ammonium acetatein water; or (iii) 0.05% TFA in water.

B channel: acetonitrile (organic phase).

The detector was UV measured at dual wavelengths: 220 and 260 nm.

The LCMS gradients were one of the following:

1. LCMS reaction monitoring and final compound analysis method (forgeneral polarity compounds)

Gradient condition: 5 min run time

Time Programs: P1:10 mM ammonium acetate in water/acetonitrile

Q1: 0.05% TFA in water/acetonitrile,

R1: 0.05% formic acid in water/acetonitrile.

The gradient varied acetonitrile from 10% to 90% to 10%.

Flow rate: 1.2 mL/min.

2. LCMS reaction monitoring and final compound analysis method in 12 minrun (for close eluting compounds):

Gradient condition: 12 min run time

Time Programs: P2: 10 mM ammonium acetate in water/acetonitrile

Q2: 0.05% TFA in water/acetonitrile

R2: 0.05% formic acid in water/acetonitrile

The gradient varied acetonitrile from 5% to 90% to 5%

Flow rate: 1.0 mL/min.

3. LCMS after method development in HPLC—gradient conditions are as perHPLC.

Mass spectral data was obtained using the following:

Ionization technique: ESI (Electron Spray Ionization) using API(Atmospheric pressure Ionization) source.

Declustering Potential: 10-70 V depending on the ionization of compound

Mass range: 100-800 amu

Scan type: Q1

Polarity: +/−ve

Ion Source: Turbo spray

Ion spray voltage: +5500 for +ve mode and −4500 for −ve mode

Mass Source temperature: 200° C.

Synthesis of Intermediates Used in Producing Compounds of Formula (I).

The following eighteen schemes show the synthesis of certainintermediates used to make compounds of formula (I). It will be apparentand understood by one of ordinary skill in the art that the chemicalstructures represented below that contain heteroatoms may, in certaincircumstances, have one or more bound hydrogens that are not shown.

Step 1: Preparation of benzofuran-5-carbaldehyde (B)

To an ice-cold stirred solution of compound A (500 mg, 3.37 mmol, 1 eq)in chlorobenzene (10 ml) were added NBS (721 mg, 4.05 mmol, 1.2 eq) inportions and AIBN (11 mg, 0.07 mmol, 0.02 eq) and the resulting solutionwas stirred at 80° C. for 4 h. The reaction mixture was cooled to RT,concentrated in vacuo and the residue was washed with saturated aq.NaHCO₃ solution (20 ml). The organic components were extracted withethyl acetate (50 ml) and the ethyl acetate layer was concentrated invacuo. The crude material was purified by flash chromatography(Combiflash) using 100-200 mesh silica gel eluting with 5% ethylacetate/hexane to obtain the compound B (270 mg, 55%) as white solid.

¹H NMR (400 MHz, DMSO-d₆) δ 10.06 (s, 1H), 8.28 (s, 1H), 8.16-8.16 (m,1H), 7.89-7.87 (m, 1H), 7.81-7.79 (m, 1H), 7.16-7.15 (m, 1H);

LCMS: m/z=147.4 [M+H], RT=2.99 minutes; (Program R1, Column Y).

Step 2: Preparation of 1-benzofuran-5-yl-ethanol (C)

To a stirred solution of compound B (900 mg, 6.16 mmol, 1 eq) in dry THF(20 mL) was added methyl magnesium bromide (3 M in diethyl ether, 4.1mL, 12.3 mmol, 2 eq) drop wise at −50° C. and the resulting mixture wasstirred for 4 h at −50° C. The reaction mixture was quenched withsaturated aq. NH₄Cl solution (20 ml) and the organic components wereextracted with ethyl acetate (200 ml). The ethyl acetate layer was driedover anhydrous sodium sulfate, concentrated in vacuo to obtain compoundC (900 mg, 90%) as colorless sticky material which was used in the nextstep without further purification.

¹H NMR (400 MHz, DMSO-d₆) δ 7.97-7.94 (m, 1H), 7.60 (s, 1H), 7.50 (d,J=8 Hz, 1H), 7.29-7.27 (m, 1H), 6.94-6.91 (m, 1H), 5.15 (d, J=8 Hz, 1H),4.84-4.78 (m, 1H), 1.35 (d, J=8 Hz, 3H).

Step 3: Preparation of 5-(1-azido-ethyl)-benzofuran (D)

To a stirred solution of compound C (200 mg, 1.23 mmol, 1 eq) in drytoluene (10 mL) were added DPPA (408 mg, 1.5 mmol, 1.2 eq) and DABCO(168 mg, 1.5 mmol, 1.2 eq) at 0° C. and the resulting mixture wasstirred at 23° C. for 18 h. The reaction mixture was concentrated invacuo. The crude residue was purified by flash chromatography(Combiflash) using 100-200 mesh silica gel and eluting with 20% ethylacetate/hexane to obtain the compound D (40 mg, 18%) as brown stickymaterial.

¹H NMR (400 MHz, DMSO-d₆) δ 8.02-8.02 (m, 1H), 7.69 (s, 1H), 7.62 (d,J=8 Hz, 1H), 7.34 (d, J=8 Hz, 1H), 6.98-6.98 (m, 1H), 4.96-4.91 (m, 1H),1.50 (d, J=4 Hz, 3H).

Step 4: Preparation of 1-benzofuran-5-yl-ethylamine (E)

To a stirred solution of compound D (40 mg, 0.21 mmol, 1 eq) in amixture of THF (5 mL) and H₂O (1 ml) was added triphenylphosphine (111mg, 0.42 mmol, 2 eq) and the resulting mixture was stirred at 70° C. for18 h. The reaction mixture was cooled to RT and concentrated in vacuo.The crude residue was purified by flash chromatography (Combiflash)using 100-200 mesh silica gel and eluting with 10%methanol/dichloromethane to obtain the compound E (30 mg, 88%) as brownsticky material.

¹H NMR (400 MHz, DMSO-d₆) δ 7.95 (d, J=4 Hz, 1H), 7.63 (s, 1H), 7.50 (d,J=8 Hz, 1H), 7.33-7.31 (m, 1H), 6.91-6.91 (m, 1H), 4.16-4.11 (m, 1H),1.30 (d, J=8 Hz, 3H);

LCMS: m/z=162.2 [M+H], RT=1.56 minutes; (Program R1, Column Y).

Step 1: Preparation of 4-prop-2-ynyloxy-benzaldehyde (C)

To a stirred solution of compound A (5.91 g, 48.4 mmol, 1 eq) in toluene(80 mL) were added compound B (80 wt. % in toluene, 8 mL, 89.8 mmol,1.85 eq) and K₂CO₃ (87.4 g, 633 mmol, 13 eq) at 0° C. and the resultingmixture was stirred at 100° C. for 8 h. The reaction mixture was cooledto RT, filtered and the filtrate was concentrated in vacuo. The crudematerial was purified by flash chromatography (Combiflash) using 100-200mesh silica gel eluting with 20% ethyl acetate/hexane to obtain thecompound C (3 g, 40%) as white solid.

¹H NMR (400 MHz, DMSO-d₆) δ 9.88 (s, 1H), 7.89 (d, J=8 Hz, 2H), 7.17 (d,J=8 Hz, 2H), 4.94 (d, J=2 Hz, 2H), 3.64 (br s, J=4 Hz, 1H);

LCMS: m/z=161.2 [M+H], RT=2.96 minutes; (Program R1, Column Y).

Step 2: Preparation of 2-methyl-benzofuran-5-carbaldehyde (D)

To compound C (3 g, 18.75 mmol, 1 eq) was added PEG-300 (30 mL) and theresulting mixture was heated to 220° C. for 4 h. The reaction mixturewas cooled to RT, diluted with ice-water (70 ml) and the organiccomponents were extracted with ethyl acetate (200 ml). The ethyl acetatelayer was concentrated in vacuo and the crude material was purified byflash chromatography (Combiflash) using 100-200 mesh silica gel elutingwith 20% ethyl acetate/hexane to obtain the compound D (700 mg, 24%) aswhite solid.

¹H NMR (400 MHz, DMSO-d₆) δ 10.02 (s, 1H), 8.12 (s, 1H), 7.80-7.78 (m,1H), 7.69-7.67 (m, 1H), 6.76 (s, 1H), 2.48 (s, 3H);

LCMS: m/z=161.2 [M+H], RT=3.22 minutes; (Program R1, Column Y).

Step 3: Preparation of 1-(2-methyl-benzofuran-5-yl)-ethanol (E)

To a stirred solution of compound D (700 mg, 4.37 mmol, 1 eq) in dry THF(15 mL) was added methyl magnesium bromide (3M in diethyl ether, 3 mL,8.75 mmol, 2 eq) drop wise at −50° C. and the resulting mixture wasstirred for 4 h at −50° C. The reaction mixture was quenched withsaturated aqueous NH₄Cl solution (20 ml) and the organic components wereextracted with ethyl acetate (50 ml). The ethyl acetate layer wasconcentrated in vacuo to obtain compound E (570 mg, 74%) as colorlesssticky material.

¹H NMR (400 MHz, DMSO-d₆) δ 7.45 (s, 1H), 7.38 (d, J=8 Hz, 1H),7.19-7.16 (m, 1H), 6.52 (s, 1H), 5.11 (d, J=4 Hz, 1H), 4.79-4.76 (m,1H), 2.42 (s, 3H), 1.34 (d, J=4 Hz, 3H);

LCMS: m/z=175.0 [M−H], RT=3.03 minutes; (Program R1, Column Y).

Step 4: Preparation of 5-(1-azido-ethyl)-2-methyl-benzofuran (F)

To a stirred solution of compound E (600 mg, 3.41 mmol, 1 eq) in drytoluene (10 mL) were added DPPA (1.12 g, 4.09 mmol, 1.2 eq) and DABCO(459 mg, 4.09 mmol, 1.2 eq) at 0° C. and the resulting mixture wasstirred at 23° C. for 18 h. The reaction mixture was concentrated invacuo and the crude was purified by flash chromatography (Combiflash)using 100-200 mesh silica gel eluting with 10% ethyl acetate/hexane toobtain the compound F (210 mg, 31%) as brown sticky material.

¹H NMR (400 MHz, DMSO-d₆) δ 7.54 (s, 1H), 7.49 (d, J=8 Hz, 1H),7.24-7.22 (m, 1H), 6.59 (s, 1H), 4.90-4.88 (m, 1H), 2.44 (s, 3H), 1.48(d, J=8 Hz, 3H).

Step 5: Preparation of 1-(2-methyl-benzofuran-5-yl)-ethylamine (G)

To a stirred solution of compound F (210 mg, 1.04 mmol, 1 eq) in amixture of THF (20 mL) and H₂O (4 ml) was added triphenylphosphine (550mg, 2.09 mmol, 2 eq) and the resulting mixture was stirred at 70° C. for18 h. The reaction mixture was cooled to RT and concentrated in vacuo.The crude material was purified by flash chromatography (Combiflash)using 100-200 mesh silica gel eluting with 10% methanol/dichloromethaneto obtain the compound G (130 mg, 77%) as brown sticky material.

¹H NMR (400 MHz, DMSO-d₆) δ 7.48 (s, 1H), 7.36 (d, J=8 Hz, 1H),7.21-7.19 (m, 1H), 6.51 (s, 1H), 4.08-4.03 (m, 1H), 2.41 (s, 3H), 2.15(br, 2H), 1.26 (d, J=8 Hz, 3H);

LCMS: m/z=176.1 [M+H], RT=2.06 minutes; (Program R1, Column Y).

Step 1: Preparation of 1-(4-Isopropoxy-phenyl)-ethanol (B)

To a stirred solution of compound A (1 g, 6.09 mmol, 1 eq) in dry THF(20 mL) was added methyl magnesium bromide (3 M in diethyl ether, 4 mL,12.18 mmol, 2 eq) drop wise at −50° C. and stirred for 4 h at −50° C.The reaction mixture was quenched with saturated aq. NH₄Cl solution (30ml) and the organic components were extracted with ethyl acetate (100ml). The solvent was finally removed in vacuo to obtain the compound B(960 mg, 88%) as colorless sticky material.

¹H NMR (400 MHz, DMSO-d₆) δ 7.21 (d, J=8 Hz, 2H), 6.83 (d, J=8 Hz, 2H),4.99 (d, J=4 Hz, 1H), 4.66-4.60 (m, 1H), 4.58-4.52 (m, 1H), 1.28 (d, J=8Hz, 3H), 1.24 (d, J=4 Hz, 6H);

LCMS: m/z=163.1 [M+H], RT=3.40 minutes; (Program R1, Column Y).

Step 2: Preparation of 1-(1-Azido-ethyl)-4-isopropoxy-benzene (C)

To a stirred solution of compound B (450 mg, 2.5 mmol, 1 eq) in drytoluene (10 mL) was added DPPA (826 mg, 3 mmol, 1.2 eq) and DABCO (337mg, 3 mmol, 1.2 eq) respectively at 0° C. and the reaction mixture wasstirred at 23° C. for 18 h. The reaction mixture was concentrated invacuo and the crude was purified by flash chromatography (Combiflash)using 100-200 mesh silica gel eluting with 10% ethylacetate/hexane toobtain the compound C (240 mg, 47%) as brown sticky material.

¹H NMR (400 MHz, DMSO-d₆) δ 7.28 (d, J=8 Hz, 1H), 7.15-7.12 (m, 1H),6.92-6.79 (m, 2H), 4.77-4.72 (m, 1H), 4.65- (m, 1H), 1.42 (d, J=8 Hz,3H), 1.33 (d, J=8 Hz, 6H).

Step 3: Preparation of 1-(4-Isopropoxy-phenyl)-ethylamine (D)

To a stirred solution of compound C (240 mg, 1.17 mmol, 1 eq) in THF (20mL) and H₂O (4 ml) mixture, was added triphenylphosphine (615 mg, 2.34mmol, 2 eq) and stirred at 70° C. for 18 h. The reaction mixture wasconcentrated in vacuo and the crude was purified by flash chromatography(Combiflash) using 100-200 mesh silica gel eluting with 10%methanol/dichloromethane to obtain the compound G (90 mg, 43%) as brownsticky material.

¹H NMR (400 MHz, DMSO-d₆) δ 7.23 (d, J=8 Hz, 2H), 6.81 (d, J=8 Hz, 2H),4.57-4.51 (m, 1H), 3.94-3.89 (m, 1H), 2.1 (br s, 2H), 1.24-1.19 (m, 9H);

LCMS: m/z=180.3 [M+H], RT=2.06 minutes; (Program R1, Column Y).

Step 1: Preparation of 4-hydroxy-3-iodo-benzaldehyde (B)

To a stirred solution of compound A (2 g, 16.4 mmol, 1 eq) in AcOH (30mL) was added NIS (4.5 g, 19.7 mmol, 1.2 eq) in portions and theresulting mixture was stirred for 18 h at 23° C. The reaction mixturewas filtered and the filtrate was diluted with ethyl acetate (100 mL).The ethyl acetate layer was washed with water (30 mL) and concentratedin vacuo to obtain the compound B (3.2 g, 79%) as white solid which wasused in the next step without further purification.

¹H NMR (400 MHz, DMSO-d₆) δ 11.51 (s, 1H), 11.1 (br s, 1H), 8.23 (s,1H), 7.02 (d, J=8 Hz, 1H), 6.92 (d, J=8 Hz, 1H);

LCMS: m/z=249.2 [M+H], RT=3.23 minutes; (Program R1, Column Y).

Step 2: Preparation of 3-iodo-4-methoxymethoxy-benzaldehyde (C)

To a stirred solution of compound B (3.2 g, 12.9 mmol, 1 eq) in dry DMF(15 mL) were added K₂CO₃ (7.12 g, 51.6 mmol, 4 eq) and MOM chloride (1.3g, 15.5 mmol, 1.2 eq) in portions at 0° C. and the resulting mixture wasstirred for 16 h at 23° C. The reaction mixture was diluted withice-water (30 ml) and the organic components were extracted with ethylacetate (100 ml). The organic layer was concentrated in vacuo and thecrude material was purified by flash chromatography (Combiflash) using100-200 mesh silica gel eluting with 20% ethylacetate/hexane to obtainthe compound C (1 g, 27%) as white solid.

¹H NMR (400 MHz, DMSO-d₆) δ 9.83 (s, 1H), 8.31 (d, J=4 Hz, 1H),7.91-7.88 (m, 1H), 7.27 (d, J=8 Hz, 1H), 5.40 (s, 2H), 3.41 (s, 3H);

LCMS: m/z=293.0 [M+H], RT=3.71 minutes; (Program R1, Column Y).

Step 3: Preparation of3-cyclopropylethynyl-4-methoxymethoxy-benzaldehyde (E)

To a solution of compound C (200 mg, 0.68 mmol, 1 eq) in triethylamine(2 mL) in sealed tube were added compound D (50 mg, 0.75 mmol, 1.1 eq)and CuI (3 mg, 0.013 mmol, 0.02 eq) the mixture was degassed with argonfor 15 minutes. PdCl₂(Ph₃P)₂ (10 mg, 0.013 mmol, 0.02 eq) was added toit and the resulting mixture was degassed with argon for 10 minutes andstirred at 50° C. for 18 h. The reaction mixture was cooled to RT,filtered and the filtrate was diluted with ethyl acetate (20 ml). Theorganic layer was washed with water (10 ml) and concentrated in vacuo.The crude material was purified by flash chromatography (Combiflash)using 100-200 mesh silica gel eluting with 10% ethyl acetate/hexane toobtain the compound E (140 mg, 87%) as white solid.

¹H NMR (400 MHz, DMSO-d₆) δ 9.83 (s, 1H), 7.87 (d, J=4 Hz, 1H),7.73-7.70 (m, 1H), 7.19 (d, J=8 Hz, 1H), 5.30 (s, 2H), 3.51 (s, 3H),1.53-1.46 (m, 1H), 0.92-0.87 (m, 2H), 0.85-0.82 (m, 2H);

LCMS: m/z=231.2 [M+H], RT=3.77 minutes; (Program R1, Column Y).

Step 4: Preparation of 2-cyclopropyl-benzofuran-5-carbaldehyde (F)

To a stirred solution of compound E (2.7 g, 11.74 mmol 1 eq) in amixture of iPrOH (30 mL) and THF (30 mL) was added 6N aqueous HCl (30mL) drop wise at 0° C. and the resulting mixture was stirred for 18 h at23° C. The reaction mixture was concentrated in vacuo and the residuewas diluted with ethyl acetate (100 ml). The ethyl acetate layer waswashed with water (30 ml) and concentrated in vacuo. The crude materialwas purified by flash chromatography (Combiflash) using 100-200 meshsilica gel eluting with 20% ethylacetate/hexane to obtain the compound F(1 g, 46%) as white solid

¹H NMR (400 MHz, DMSO-d₆) δ 10.01 (s, 1H), 8.08 (s, 1H), 7.78-7.76 (m,1H), 7.65 (d, J=8 Hz, 1H), 6.75 (s, 1H), 2.20-2.13 (m, 1H), 1.10-1.00(m, 2H), 0.98-0.95 (m, 2H).

Step 5: Preparation of 1-(2-cyclopropyl-benzofuran-5-yl)-ethanol (G)

To a stirred solution of compound F (1 g, 5.38 mmol, 1 eq) in dry THF(20 mL) was added methyl magnesium bromide (3M in diethyl ether 3.6 mL,10.75 mmol, 2 eq) drop wise at −50° C. and the resulting mixture wasstirred for 4 h at −50° C. The reaction mixture was quenched withsaturated aq. NH₄Cl solution and the organic components were extractedwith ethyl acetate (100 ml). The ethyl acetate layer was concentrated invacuo to obtain the compound G (980 mg, 91%) as colorless stickymaterial which was used in the next step without further purification.

¹H NMR (400 MHz, DMSO-d₆) δ 7.42 (s, 1H), 7.35 (d, J=8 Hz, 1H),7.16-7.14 (m, 1H), 6.52 (s, 1H), 5.093 (d, J=4 Hz, 1H), 4.79-4.74 (m,1H), 2.12-2.05 (m, 1H), 1.329 (d, J=6 Hz, 3H), 1.01-0.96 (m, 2H),0.87-0.84 (m, 2H);

LCMS: m/z=202.2 [M+H], RT=3.65 minutes; (Program R1, Column Y).

Step 6: Preparation of 5-(1-azido-ethyl)-2-cyclopropyl-benzofuran (H)

To a stirred solution of compound G (200 mg, 0.99 mmol, 1 eq) in drytoluene (5 mL) were added DPPA (327 mg, 1.19 mmol, 1.2 eq) and DABCO(134 mg, 1.19 mmol, 1.2 eq) at 0° C. for 2 h followed by at 23° C. 18 h.The reaction mixture was concentrated in vacuo and the crude materialwas purified by flash chromatography (Combiflash) using 100-200 meshsilica gel eluting with 10% ethyl acetate/hexane to obtain the compoundH (40 mg, 18%) as colorless sticky material.

¹H NMR (400 MHz, DMSO-d₆) δ 7.51-7.45 (m, 2H), 7.33-7.32 (m, 1H),7.22-7.20 (m, 1H), 4.89-4.88 (m, 1H), 1.47 (d, J=8 Hz, 3H), 1.29-1.27(m, 1H), 1.01-0.99 (m, 2H), 0.89-0.88 (m, 2H).

Step 7: Preparation of 1-(2-cyclopropyl-benzofuran-5-yl)-ethylamine (I)

To a stirred solution of compound H (40 mg, 0.18 mmol, 1 eq) in amixture of THF (50 mL) and H₂O (1 ml) was added triphenylphosphine (93mg, 0.35 mmol, 2 eq) and the resulting mixture was stirred at 70° C. for18 h. The reaction mixture was cooled to RT, concentrated in vacuo andthe crude was purified by flash chromatography (Combiflash) using100-200 mesh silica gel eluting with 10% methanol/dichloromethane toobtain the compound I (25 mg, 72%) as brown sticky material.

¹H NMR (400 MHz, DMSO-d₆) δ 7.45 (s, 1H), 7.33 (d, J=8 Hz, 1H),7.19-7.17 (m, 1H), 6.51 (s, 1H), 4.08-4.03 (m, 1H), 2.11-2.06 (m, 1H),1.26 (d, J=8 Hz, 3H), 1.01-0.96 (m, 2H), 0.87-0.83 (m, 2H);

LCMS: m/z=202.4 [M+H], RT=2.72 minutes; (Program R1, Column Y).

Step 1: Preparation of 6-fluoro-quinoline-3-carboxylic Acid Ethyl Ester(C)

To a stirred solution of compound A (3 g, 17.74 mmol, 1 eq) in EtOH (100mL) were added SnCl₂.2H₂O (48 g, 212.9 mmol, 12 eq) and compound B (8.43g, 44.34 mmol, 2.5 eq) drop wise and the resulting mixture was stirredat 70° C. for 12 h. The reaction mixture was concentrated in vacuo andthe residue was diluted with water. The aqueous part was further dilutedwith saturated aq. NaHCO₃ solution (100 ml) and filtered. Organiccomponents were extracted with ethyl acetate (300 ml) and the ethylacetate layer was dried over anhydrous sodium sulfate and removed invacuo. The crude residue was purified by flash chromatography(Combiflash) using 100-200 mesh silica gel and eluting with 30% ethylacetate/hexane to obtain compound C (2.6 g, 67%) as white solid.

¹H NMR (400 MHz, DMSO-d₆) δ 9.29 (s, 1H), 9.01 (s, 1H), 8.20-8.16 (m,1H), 8.07-8.04 (m, 1H), 7.87-7.82 (m, 1H), 4.42 (q, J=8 Hz, 2H), 1.38(t, J=8 Hz, 3H);

LCMS: m/z=219.8 [M+H], RT=3.66 minutes; (Program R1, Column Y).

Step 2: Preparation of 6-fluoro-quinoline-3-carboxylic Acid (D)

To a stirred solution of compound C (2.6 g, 11.9 mmol, 1 eq) in THF (60mL) was added LiOH (1.5 g, 35.6 mmol, 3 eq) in water (10 mL) drop wiseat 0° C. and the resulting mixture was stirred for 4 h at 23° C. Thereaction mixture was concentrated in vacuo and the residue was dilutedwith water. The aqueous part was washed with ethyl acetate (20 mL) andthen acidified with saturated aq. citric acid solution to pH 5. Theorganic components were extracted with ethyl acetate (200 mL), ethylacetate layer was dried over anhydrous sodium sulfate and concentratedin vacuo to obtain the compound D (2.2 g, 97%) as gummy material whichwas used in the next step without further purification.

¹H NMR (400 MHz, DMSO-d₆) δ 9.28 (d, J=2 Hz, 1H), 8.97 (d, J=2 Hz, 1H),8.19-8.15 (m, 1H), 8.04-8.01 (m, 1H), 7.85-7.79 (m, 1H);

LCMS: m/z=192.1 [M+H], RT=2.69 minutes; (Program R1, Column Y).

Step 3: Preparation of 6-fluoro-quinoline-3-carbonyl Chloride (E)

To a stirred solution of compound D (2.2 g, 11.5 mmol, 1 eq) in dryCH₂Cl₂ (100 mL) was added oxalyl chloride (8.77 g, 69.1 mmol, 6 eq) dropwise at 0° C. and the resulting mixture was stirred for 4 h at 23° C.The reaction mixture was concentrated in vacuo under nitrogen atmosphereto obtain the compound E (2.4 g) as colorless liquid which was used inthe next step without further purification.

Step 4: Preparation of 6-fluoro-quinoline-3-carboxylic Acidmethoxy-methyl-amide (F)

To a stirred solution of compound E (2.4 g, 11.5 mmol, 1 eq) in dryCH₂Cl₂ (100 mL) were added N,O-dimethylhydroxylamine hydrochloride (1.7g, 17.2 mmol, 1.5 eq) and DIPEA (14.8 g, 114.8 mmol, 10 eq) drop wise at0° C. and the resulting mixture was stirred for 18 h at 23° C. Thereaction mixture was concentrated in vacuo and the residue was dilutedwith CH₂Cl₂ (200 ml). DCM layer was washed with water (50 ml) andconcentrated in vacuo. The crude material was purified by flashchromatography (Combiflash) using 100-200 mesh silica gel eluting with60% ethyl acetate/hexane to obtain the compound F (650 mg, 25%) as offwhite solid.

¹H NMR (400 MHz, DMSO-d₆) δ 9.03 (d, J=2 Hz, 1H), 8.66 (d, J=4 Hz, 1H),8.16-8.12 (m, 1H), 7.95-7.92 (m, 1H), 7.80-7.75 (m, 1H), 3.65 (s, 3H),3.35 (s, 3H);

LCMS: m/z=235.2 [M+H], RT=3.02 minutes; (Program R1, Column Y).

Step 5: Preparation of cyclopropyl-(6-fluoro-quinolin-3-yl)-methanone(G)

To a stirred solution of compound F (650 mg, 2.78 mmol, 1 eq) in dry THF(20 mL) was added cyclopropyl magnesium bromide (0.5 M in THF, 8.5 mL,4.2 mmol, 1.5 eq) drop wise at −78° C. and the resulting mixture wasstirred for 40 h at 23° C. The reaction mixture was quenched withsaturated aq. NH₄Cl solution and the organic components were extractedwith ethyl acetate (50 ml). Ethyl acetate layer was concentrated invacuo and the crude material was purified by flash chromatography(Combiflash) using 100-200 mesh silica gel eluting with 50% ethylacetate/hexane to obtain the compound G (130 mg, 22%) as colorlesssticky material

¹H NMR (400 MHz, DMSO-d₆) δ 9.36 (d, J=2 Hz, 1H), 9.16 (d, J=2 Hz, 1H),8.20-8.16 (m, 1H), 8.01-7.98 (m, 1H), 7.87-7.82 (m, 1H), 1.821.75 (m,1H), 1.18-1.15 (m, 4H);

LCMS: m/z=215.9 [M+H], RT=3.51 minutes; (Program R1, Column Y).

Step 6: Preparation ofcyclopropyl-C-(6-fluoro-quinolin-3-yl)-methylamine (H)

To a stirred solution of compound G (130 mg, 0.6 mmol, 1 eq) inmethanolic ammonia (7 N, 10 mL) was added titanium isopropoxide (344 mg,1.2 mmol, 2 eq) drop wise at 0° C. and the mixture was stirred for 12 hat 23° C. Sodium borohydride (35 mg, 0.9 mmol, 1.5 eq) was added insmall portions to it at 0° C. and the resulting mixture was stirred for18 h at 23° C. The reaction mixture was quenched with ice and wasconcentrated in vacuo. The crude material was purified by flashchromatography (Combiflash) using 100-200 mesh silica gel eluting with10% methanol/dichloromethane to obtain the compound H (40 mg, 31%) asbrown sticky material.

¹H NMR (400 MHz, DMSO-d₆) δ 8.96 (d, J=2 Hz, 1H), 8.27-8.29 (m, 1H),8.07-8.03 (m, 1H), 7.76-7.73 (m, 1H), 7.63-7.58 (m, 1H), 4.40 (m, 1H),2.20 (br s, 2H), 0.53-0.51 (m, 1H), 0.43-0.40 (m, 4H);

LCMS: m/z=217.2 [M+H], RT=2.00 minutes; (Program R1, Column Y).

Step 1: Preparation of 1H-indazole-3-carboxylic Acidmethoxy-methyl-amide (C)

To a stirred solution of compound A (1 g, 6.2 mmol, 1 eq) in CH₂Cl₂ (30mL) were added compound B (903 mg, 9.25 mmol, 1.5 eq), HOBT (1 g, 7.4mmol, 1.2 eq) and EDCI (2.4 g, 12.3 mmol, 2 eq) at 0° C. DIPEA (4 g,30.8 mmol, 5 eq) was added to it at 0° C. and the resulting mixture wasstirred for 18 h at 23° C. The reaction mixture was diluted with CH₂Cl₂(100 ml) and DCM layer was washed with water (30 ml). DCM layer wasremoved in vacuo and the crude residue was purified by flashchromatography (Combiflash) using 100-200 mesh silica gel and elutingwith 50% ethyl acetate/hexane to obtain the compound C (700 mg, 56%) asoff white solid.

¹H NMR (400 MHz, DMSO-d₆) δ 13.60 (s, 1H) 8.00 (d, J=8 Hz, 1H), 7.61 (d,J=8 Hz, 1H), 7.41 (t, J=8 Hz, 1H), 7.23 (t, J=8 Hz, 1H), 3.78 (s, 3H),3.45 (s, 3H);

LCMS: m/z=206.2 [M+H], RT=2.55 minutes; (Program R1, Column W).

Step 2: Preparation of 1-(1H-indazol-3-yl)-ethanone (D)

To a stirred solution of compound C (700 mg, 3.4 mmol, 1 eq) in dry THF(40 mL) was added methyl magnesium bromide (1 M in diethyl ether, 11 mL,10.2 mmol, 3 eq) drop wise at 0° C. and the resulting mixture wasstirred for 18 h at 23° C. The reaction mixture was quenched withsaturated aq. NH₄Cl solution and the organic components were extractedwith ethyl acetate (100 ml). Ethyl acetate layer was concentrated invacuo and the crude material was purified by flash chromatography(Combiflash) using 100-200 mesh silica gel eluting with 40% ethylacetate/hexane to obtain the compound D (250 mg, 44%) as colorlesssticky material.

¹H NMR (400 MHz, DMSO-d₆) δ 13.82 (s, 1H), 8.17 (d, J=8 Hz, 1H), 7.66(d, J=8 Hz, 1H), 7.47-7.43 (m, 1H), 7.31 (t, J=8 Hz, 1H), 2.63 (s, 3H);

LCMS: m/z=161.1 [M+H], RT=2.94 minutes; (Program R1, Column W).

Step 3: Preparation of 1-(1H-indazol-3-yl)-ethylamine (E)

To a stirred solution of compound D (250 mg, 1.56 mmol, 1 eq) inmethanolic ammonia (7 N, 10 mL) was added titanium isopropoxide (890 mg,3.12 mmol, 2 eq) drop wise at 0° C. and the mixture was stirred for 6 hat 23° C. Sodium borohydride (90 mg, 2.34 mmol, 1.5 eq) was added to itin small portions at 0° C. and the resulting mixture was stirred for 18h at 23° C. The reaction mixture was quenched with ice and concentratedin vacuo. The crude material was purified by flash chromatography(Combiflash) using 100-200 mesh silica gel eluting with 10%methanol/dichloromethane to obtain the compound E (100 mg, 40%) as brownsticky material

¹H NMR (400 MHz, DMSO-d₆) δ 12.61 (s, 1H), 7.93 (d, J=8 Hz, 1H), 7.44(d, J=8 Hz, 1H), 7.29 (t, J=8 Hz, 1H), 7.04 (t, J=8 Hz, 1H), 4.42-4.37(m, 1H), 1.45 (d, J=8 Hz, 3H);

LCMS: m/z=162.3 [M+H], RT=0.57 minutes; (Program R1, Column W).

Step 1: Preparation of imidazo[1,2-a]pyridine-6-carboxylic Acid EthylEster (C)

To a stirred solution of compound A (3.2 g, 19.3 mmol, 1 eq) in EtOH(300 mL) were added NaHCO₃ (32.4 g, 385.5 mmol, 20 eq) and compound B(˜55% aq. solution, 30 ml, 192.8 mmol, 10 eq) at 23° C. and theresulting mixture was heated to 70° C. for 18 h. The reaction mixturewas cooled to RT, filtered and the filtrated was concentrated in vacuoand the residue was diluted with ethyl acetate (200 ml). Ethyl acetatelayer was washed with water (50 ml), dried over anhydrous Na₂SO₄,concentrated in vacuo to obtain the compound C (3.7 g) which was used inthe next step without further purification.

LCMS: m/z=190.7 [M+H], RT=0.62 minutes; (Program R1, Column W).

Step 2: Preparation of imidazo[1,2-a]pyridine-6-carboxylic Acid (D)

To a stirred solution of compound C (3.7 g, 19.5 mmol, 1 eq) in THF (45mL) was added a solution of LiOH (2.5 g, 58.4 mmol, 3 eq) in water (5mL) drop wise at 0° C. and the resulting mixture was stirred for 18 h at23° C. The reaction mixture was concentrated in vacuo and the residuewas diluted with water. The aqueous part was washed with ethyl acetate(20 mL), acidified with saturated aq. citric acid solution to pH 5. Theorganic components were extracted from the aq. part with 10% MeOH/CH₂Cl₂(100 mL) and the organic layer was concentrated in vacuo to obtain thecompound D (3 g) as off white solid which was used in the next stepwithout further purification.

LCMS: m/z=163.1 [M+H], RT=0.40 minutes; (Program R1, Column W).

Step 3: Preparation of imidazo[1,2-a]pyridine-6-carboxylic Acidmethoxy-methyl-amide (F)

To a stirred solution of compound D (2.5 g, 15.4 mmol, 1 eq) in DMF (50mL) were added compound E (2.3 g, 23.1 mmol, 1.5 eq), triethylamine(15.6 g, 154.3 mmol, 10 eq) and T3P (˜50% in ethyl acetate, 14.8 g, 23.1mmol, 1.5 eq) at 23° C. and the resulting mixture was heated at 120° C.for 18 h. The reaction mixture was cooled to RT, quenched with water andthe organic components were extracted with 10% methanol/CH₂Cl₂. Theorganic layer was concentrated in vacuo and the crude material waspurified by flash chromatography (Combiflash) using 100-200 mesh silicagel eluting with 10% MeOH/CH₂Cl₂ to obtain the compound F (600 mg, 20%)as off white solid.

¹H NMR (400 MHz, DMSO-d₆) δ 9.03 (s, 1H), 8.06 (s, 1H), 7.64 (m, 1H),7.59 (d, J=8 Hz, 1H), 7.47-7.44 (m, 1H), 3.61 (s, 3H), 3.32 (s, 3H);

LCMS: m/z=206.2 [M+H], RT=0.54 minutes; (Program R1, Column W).

Step 4: Preparation of cyclopropyl-imidazo[1,2-a]pyridin-6-yl-methanone(G)

To a stirred solution of compound F (600 mg, 2.9 mmol, 1 eq) in dry THF(30 mL) was added cyclopropyl magnesium bromide (0.5 M in THF, 12 mL,5.8 mmol, 2 eq) drop wise at 0° C. and the resulting mixture was stirredfor 18 h at 23° C. The reaction mixture was quenched with saturated aq.NH₄Cl solution and the organic components were extracted with ethylacetate (50 ml). Ethyl acetate layer was concentrated in vacuo and thecrude material was purified by flash chromatography (Combiflash) using100-200 mesh silica gel eluting with 10% MeOH/CH₂Cl₂ to obtain thecompound G (230 mg, 43%) as colorless sticky material

¹H NMR (400 MHz, DMSO-d₆) δ 9.63 (s, 1H), 8.07 (s, 1H), 7.70 (m, 2H),7.63 (m, 1H), 1.78 (m, 1H), 0.75-0.90 (m, 4H);

LCMS: m/z=187.2 [M+H], RT=0.60 minutes; (Program R1, Column W).

Step 5: Preparation ofC-cyclopropyl-C-imidazo[1,2-a]pyridin-6-yl-methylamine (H)

To a stirred solution of compound G (230 mg, 1.2 mmol, 1 eq) inmethanolic ammonia (7 N, 10 mL) was added titanium isopropoxide (703 mg,2.47 mmol, 2 eq) drop wise at 0° C. and the mixture was stirred for 18 hat 23° C. Sodium borohydride (71 mg, 1.85 mmol, 1.5 eq) was added to itin small portions at 0° C. and the resulting mixture was stirred for 18h at 23° C. The reaction mixture was quenched with ice and concentratedin vacuo. The crude residue was purified by flash chromatography(Combiflash) using 100-200 mesh silica gel eluting with 10%methanol/dichloromethane to obtain the compound H (40 mg, 18%) as brownsticky material.

¹H NMR (400 MHz, DMSO-d₆) δ 8.52 (s, 1H), 7.92 (s, 1H), 7.56-7.54 (m,2H), 7.39-7.37 (m, 1H), 4.00-4.20 (m, 1H), 1.00-1.20 (m, 1H), 0.58 (m,1H), 0.47-0.40 (m, 2H), 0.25-0.35 (m, 1H);

LCMS: m/z=188.2 [M+H], RT=0.38 minutes; (Program R1, Column W).

Step 1: Preparation of 6-phenyl-nicotinic Acid (C)

To a stirred solution of compound A (3 g, 16.16 mmol, 1 eq) in a mixtureof dioxane and water (150 mL, 4:1) mixture were added compound B (3 g,24.2 mmol, 1.5 eq) and sodium carbonate (8.6 g, 80.8 mmol, 5 eq) and themixture was degassed with argon for 30 minutes. Pd(Ph₃P)₄ (1.9 g, 1.62mmol, 0.1 eq) was added to it and the resulting mixture was furtherdegassed with argon for another 10 minutes and stirred at 110° C. for 18h. The reaction mixture was filtered and the filtrate was concentratedin vacuo. The residue was diluted with water (30 mL) and washed withethyl acetate (20 mL). The aq. part was acidified with saturated aq.citric acid solution to pH 5. The organic components were extracted with10% MeOH/CH₂Cl₂ (100 mL) and the organic layer was concentrated in vacuoto obtain the compound C (2.2 g, 69%) as white solid which was used inthe next step without further purification.

¹H NMR (400 MHz, DMSO-d₆) δ 13.4 (br s, 1H), 9.14 (d, J=2 Hz, 1H),8.33-8.31 (m, 1H), 8.17-8.15 (m, 2H), 8.10 (d, J=8 Hz, 1H), 7.55-7.48(m, 3H);

LCMS: m/z=200.1 [M+H], RT=3.21 minutes; (Program R1, Column Y).

Step 2: Preparation of 6-phenyl-nicotinoyl Chloride (D)

To a stirred solution of compound C (2.2 g, 11.05 mmol, 1 eq) in dryCH₂Cl₂ (50 mL) was added oxalyl chloride (8.42 g, 66.33 mmol, 6 eq) dropwise at 0° C. and the resulting mixture was stirred for 4 h at 23° C.The reaction mixture was concentrated in vacuo under nitrogen atmosphereto obtain the compound D (2.4 g) as colorless liquid which was used inthe next step without further purification.

Step 3: Preparation of N-methoxy-N-methyl-6-phenyl-nicotinamide (F)

To a stirred solution of compound D (2.4 g, 11.06 mmol, 1 eq) in dryCH₂Cl₂ (100 mL) were added compound E (N,O-dimethylhydroxylaminehydrochloride) (1.62 g, 16.6 mmol, 1.5 eq) and DIPEA (14.3 g, 110.6mmol, 10 eq) drop wise at 0° C. and the resulting mixture was stirredfor 18 h at 23° C. The reaction mixture was concentrated in vacuo andthe residue was diluted with CH₂Cl₂ (200 ml). The organic layer waswashed with water (50 ml) and concentrated in vacuo to obtain thecompound F (3.1 g) which was used in the next step without furtherpurification.

¹H NMR (400 MHz, DMSO-d₆) δ 8.885-8.882 (m, 1H), 8.15-8.13 (m, 2H),8.11-8.09 (m, 1H), 8.07-8.05 (m, 1H), 7.54-7.48 (m, 3H), 3.60 (s, 3H),3.31 (s, 3H);

LCMS: m/z=243.2 [M+H], RT=3.37 minutes; (Program R1, Column Y).

Step 4: Preparation of cyclopropyl-(6-phenyl-pyridin-3-yl)-methanone (G)

To a stirred solution of compound F (1 g, 4.13 mmol, 1 eq) in dry THF(20 mL) was added cyclopropyl magnesium bromide (0.5 M in THF, 12.5 mL,6.2 mmol, 1.5 eq) drop wise at −40° C. and the resulting mixture wasstirred for 18 h at 23° C. The reaction mixture was quenched withsaturated aq. NH₄Cl solution and the organic components were extractedwith ethyl acetate (100 ml). Ethyl acetate layer was concentrated invacuo and the crude residue was purified by flash chromatography(Combiflash) using 100-200 mesh silica gel eluting with 50%ethylacetate/hexane to obtain the compound G (320 mg, 35%) as colorlesssticky material.

¹H NMR (400 MHz, DMSO-d₆) δ 9.32-9.31 (m, 1H), 8.46-8.43 (m, 1H),8.20-8.14 (m, 3H), 7.57-7.51 (m, 3H), 3.02-2.99 (m, 1H), 1.11-1.09 (m,4H);

LCMS: m/z=223.7 [M+H], RT=3.85 minutes; (Program R1, Column Y).

Step 5: Preparation of cyclopropyl-C-(6-phenyl-pyridin-3-yl)-methylamine(H)

To a stirred solution of compound G (320 mg, 1.43 mmol, 1 eq) inmethanolic ammonia (7 N, 10 mL) was added titanium isopropoxide (816 mg,2.87 mmol, 2 eq) drop wise at 0° C. and the mixture was stirred for 8 hat 23° C. Sodium borohydride (82 mg, 2.15 mmol, 1.5 eq) was added to itin small portions at 0° C. and the resulting mixture was stirred for 18h at 23° C. The reaction mixture was quenched with ice and concentratedin vacuo. The crude residue was purified by flash chromatography(Combiflash) using 100-200 mesh silica gel eluting with 10%methanol/dichloromethane to obtain the compound H (100 mg, 31%) ascolorless sticky material

¹H NMR (400 MHz, DMSO-d₆) δ 8.66 (s, 1H), 8.06 (d, J=8 Hz, 2H), 7.90 (s,2H), 7.48 (t, J=8 Hz, 2H), 7.43-7.39 (m, 1H), 4.40 (br s, 1H), 1.06-0.98(m, 1H), 0.52-0.46 (m, 1H), 0.41-0.40 (m, 3H);

LCMS: m/z=224.8 [M+H], RT=1.83 minutes; (Program R1, Column Y).

Step 1: Preparation of 6-cyclopropylmethoxy-nicotinic Acid (C)

To a stirred solution of compound A (5 g, 31.7 mmol, 1 eq) in DMSO (100mL) were added KOH (5.3 g, 95.2 mmol, 3 eq) and compound B (3.43 g, 47.6mmol, 1.5 eq) at 23° C. and the resulting mixture was heated for 40 h at100° C. The reaction mixture was cooled to RT, diluted with water (50mL) and acidified with 6 N aq. HCl to pH 4. The organic components wereextracted with ethyl acetate (100 mL) and the ethyl acetate layer wasconcentrated in vacuo to obtain the compound C (4.2 g, 69%) as off whitesolid.

¹H NMR (400 MHz, DMSO-d₆) δ 13.00 (s, 1H), 8.69 (d, J=4 Hz, 1H),8.13-8.11 (m, 1H), 6.89 (d, J=8 Hz, 1H), 4.16 (d, J=8 Hz, 2H), 1.28-1.21(m, 1H), 0.57-0.53 (m, 2H), 0.35-0.31 (m, 2H);

LCMS: m/z=194.0[M+H], RT=3.35 minutes; (Program R1, Column Y).

Step 2: Preparation of6-cyclopropylmethoxy-N-methoxy-N-methyl-nicotinamide (E)

To a stirred solution of compound C (500 mg, 2.6 mmol, 1 eq) in CH₂Cl₂(10 mL) were added compound D (380 mg, 3.89 mmol, 1.5 eq), HOBT (420 mg,3.1 mmol, 1.2 eq) and EDCI (1 g, 5.18 mmol, 2 eq) respectively at 0° C.DIPEA (1.7 g, 12.9 mmol, 1.2 eq) was added to it at 0° C. and theresulting mixture was stirred for 18 h at 23° C. The reaction mixturewas diluted with CH₂Cl₂ (50 ml) and the DCM layer was washed with water(20 ml). DCM was concentrated in vacuo and the crude material waspurified by flash chromatography (Combiflash) using 100-200 mesh silicagel eluting with 50% ethyl acetate/hexane to obtain the compound E (530mg, 87%) as off white solid

¹H NMR (400 MHz, DMSO-d₆) δ 8.45 (d, J=4 Hz, 1H), 7.96-7.93 (m, 1H),6.87 (d, J=8 Hz, 1H), 4.14 (d, J=8 Hz, 2H), 3.56 (s, 3H), 3.25 (s, 3H),1.26-1.23 (m, 1H), 0.57-0.53 (m, 2H), 0.34-0.31 (m, 2H);

LCMS: m/z=237.2 [M+H], RT=3.42 minutes; (Program R1, Column Y).

Step 3: Preparation ofcyclopropyl-(6-cyclopropylmethoxy-pyridin-3-yl)-methanone (F)

To a stirred solution of compound E (530 mg, 2.2 mmol, 1 eq) in dry THF(20 mL) was added cyclopropyl magnesium bromide (0.5 M in THF, 6.8 mL,3.37 mmol, 1.5 eq) drop wise at −78° C. and the resulting mixture wasstirred for 18 h at 23° C. The reaction mixture was quenched withsaturated aq. NH₄Cl solution and the organic components were extractedwith ethyl acetate (100 ml). The organic layer was concentrated in vacuoand the crude residue was purified by flash chromatography (Combiflash)using 100-200 mesh silica gel eluting with 40% ethyl acetate/hexane toobtain the compound F (190 mg, 39%) as colorless sticky material.

¹H NMR (400 MHz, DMSO-d₆) δ 8.93 (d, J=2 Hz, 1H), 8.25-8.22 (m, 1H),6.93 (d, J=8 Hz, 1H), 4.20 (d, J=8 Hz, 2H), 2.91-2.85 (m, 1H), 1.29-1.23(m, 1H), 1.03-1.01 (m, 4H), 0.58-0.54 (m, 2H), 0.36-0.35 (m, 2H);

LCMS: m/z=218.4 [M+H], RT=3.86 minutes; (Program R1, Column Y).

Step 4: Preparation ofC-cyclopropyl-C-(6-cyclopropylmethoxy-pyridin-3-yl)-methylamine (G)

To a stirred solution of compound F (190 mg, 0.87 mmol, 1 eq) inmethanolic ammonia (7 N, 15 mL) was added titanium isopropoxide (498 mg,1.75 mmol, 2 eq) drop wise at 0° C. and the mixture was stirred for 18 hat 23° C. Sodium borohydride (50 mg, 1.31 mmol, 1.5 eq) was added to itin small portions at 0° C. and the resulting mixture was stirred for 18h at 23° C. The reaction mixture was quenched with ice and concentratedin vacuo. The crude residue was purified by flash chromatography(Combiflash) using 100-200 mesh silica gel eluting with 10%methanol/dichloromethane to obtain the compound G (60 mg, 32%) as brownsticky material.

¹H NMR (400 MHz, DMSO-d₆) δ 8.07 (d, J=4 Hz, 1H), 7.75-7.72 (m, 1H),6.74 (d, J=8 Hz, 1H), 4.41 (br s, 2H), 4.05 (d, J=7 Hz, 2H), 3.12 (d,J=8 Hz, 1H), 1.20-1.19 (m, 1H), 0.96-0.92 (m, 1H), 0.55-0.50 (m, 2H),0.47-0.42 (m, 1H), 0.35-0.28 (m, 4H), 0.25-0.18 (m, 1H);

LCMS: m/z=219.2 [M+H], RT=2.27 minutes; (Program R1, Column Y).

Step 1: Preparation of benzo[b]thiophene-5-carbonitrile (B)

To a stirred solution of compound A (500 mg, 2.35 mmol, 1 eq) in DMF (7mL) in a sealed tube were added CuCN (273 mg, 3.05 mmol, 1.3 eq) andpyridine (0.25 mL) respectively. The resulting mixture was degassed withargon for 30 minutes and stirred for 10 h at 170° C. The reactionmixture was cooled to RT and quenched with a solution of ethylenediamine (0.25 mL) in water (6 mL) and the organic components wereextracted with ethyl acetate (60 ml). The organic layer was concentratedin vacuo and the crude material was purified by flash chromatography(Combiflash) using 100-200 mesh silica gel eluting with 10% ethylacetate/hexane to obtain the compound B (200 mg, 54%) as off whitesolid.

¹H NMR (400 MHz, DMSO-d₆) δ 8.43 (s, 1H), 8.26 (d, J=8 Hz, 1H), 7.99 (2,J=8 Hz, 1H), 7.73-7.71 (m, 1H), 7.58 (d, J=6 Hz, 1H).

Step 2: Preparation of 1-benzo[b]thiophen-5-yl-ethanone (C)

To a stirred solution of compound B (500 mg, 3.14 mmol, 1 eq) in dry THF(20 mL) was added methyl magnesium bromide (3 M in diethyl ether, 3.14mL, 9.42 mmol, 3 eq) drop wise at −78° C. and the resulting mixture wasstirred for 18 h at 23° C. The reaction mixture was quenched withsaturated aq. NH₄Cl solution and the organic components were extractedwith ethyl acetate (100 ml). The organic layer was concentrated in vacuoand the crude residue was purified by flash chromatography (Combiflash)using 100-200 mesh silica gel eluting with 40% ethyl acetate/hexane toobtain the compound F (45 mg, 8%) as colorless sticky material.

¹H NMR (400 MHz, DMSO-d₆) δ 8.55 (d, J=1 Hz, 1H), 8.13 (d, J=8 Hz, 1H),7.92-7.88 (m, 2H), 7.62 (d, J=6 Hz, 1H), 2.65 (s, 3H).

Step 3: Preparation of 1-benzo[b]thiophen-5-yl-ethylamine (D)

To a stirred solution of compound C (300 mg, 1.70 mmol, 1 eq) inmethanolic ammonia (7 N, 10 mL) was added titanium isopropoxide (970 mg,3.41 mmol, 2 eq) drop wise at 0° C. and the mixture was stirred for 5 hat 23° C. Sodium borohydride (78 mg, 2.04 mmol, 1.2 eq) was added to itin small portions at 0° C. and the resulting mixture was stirred for 18h at 23° C. The reaction mixture was quenched with ice and concentratedin vacuo. The crude residue was purified by flash chromatography(Combiflash) using 100-200 mesh silica gel eluting with 10%methanol/dichloromethane to obtain the compound G (50 mg, 17%) as brownsticky material.

¹H NMR (400 MHz, DMSO-d₆) δ 7.91 (d, J=8 Hz, 1H), 7.85 (s, 1H), 7.72 (d,J=8 Hz, 1H), 7.42-7.38 (m, 2H), 4.15-4.13 (m, 1H), 2.82 (br s, 2H), 1.31(d, J=8 Hz, 3H);

LCMS: m/z=178.2 [M+H], RT=2.09 minutes; (Program R1, Column Y).

Step 1: Preparation of 1-(4-ethoxy-phenyl)-ethanol (B)

To a stirred solution of compound A (500 mg, 3.33 mmol, 1 eq) in dry THF(20 mL) was added methyl magnesium bromide (3 M in diethyl ether, 3.33mL, 9.99 mmol, 3 eq) drop wise at −78° C. and the resulting mixture wasstirred for 3 h at 23° C. The reaction mixture was quenched withsaturated aq. NH₄Cl solution and the organic components were extractedwith ethyl acetate (60 ml). Ethyl acetate layer was concentrated invacuo and the crude residue was purified by flash chromatography(Combiflash) using 100-200 mesh silica gel eluting with 3% ethylacetate/hexane to obtain the compound F (400 mg) as colorless stickymaterial.

¹H NMR (400 MHz, DMSO-d₆) δ 7.22 (d, J=8 Hz, 2H), 6.84 (d, J=8 Hz, 2H),4.99 (d, J=4 Hz, 1H), 4.67-4.61 (m, 1H), 4.01-3.96 (m, 2H), 1.32-1.27(m, 6H).

Step 2: Preparation of methanesulfonic acid 1-(4-ethoxy-phenyl)-ethylEster (C)

To a stirred solution of compound B (400 mg, 2.4 mmol, 1 eq) in dryCH₂Cl₂ (15 mL) were added triethyl amine (0.5 mL, 3.61 mmol, 1.5 eq)followed by methane sulphonyl chloride (0.3 mL, 3.61 mmol, 1.5 eq) dropwise at 0° C. and the resulting mixture was stirred for 14 h at 23° C.The reaction mixture was quenched with water and the organic componentswere extracted with CH₂Cl₂ (50 ml). DCM layer was concentrated in vacuoto obtain the compound C (600 mg) as brownish sticky material which wasused in the next step without further purification.

Step 3: Preparation of 1-(1-azido-ethyl)-4-ethoxy-benzene (D)

To a stirred solution of compound C (600 mg, 2.45 mmol, 1 eq) in dry DMF(7 mL) was added sodium azide (1.9 g, 28.7 mmol, 12 eq) portion wise at0° C. and the resulting mixture was stirred for 14 h at 23° C. Thereaction mixture was quenched with ice and the organic components wereextracted with ethyl acetate (100 ml). Ethyl acetate was concentrated invacuo to obtain the compound D (600 mg) as brownish sticky materialwhich was used in the next step without further purification.

Step 4: Preparation of 1-(4-Ethoxy-phenyl)-ethylamine (E)

To a stirred solution of compound D (600 mg, 3.11 mmol, 1 eq) in amixture of THF (20 mL) and H₂O (2 ml) was added triphenylphosphine (1.62g, 6.22 mmol, 2 eq) and the resulting mixture was stirred at 70° C. for18 h. The reaction mixture was concentrated in vacuo and the cruderesidue was purified by flash chromatography (Combiflash) using 100-200mesh silica gel eluting with 10% methanol/dichloromethane to obtain thecompound E (30 mg) as brown sticky material.

¹H NMR (400 MHz, DMSO-d₆) δ 7.35 (d, J=8 Hz, 2H), 6.93 (d, J=8 Hz, 2H),4.26-4.21 (m, 1H), 4.04-3.99 (m, 2H), 1.40 (d, J=8 Hz, 3H), 1.35-1.29(m, 3H).

Step 1: Preparation of C-[1-(4-chloro-phenyl)-cyclobutyl]-methylamine(B)

To a stirred solution of compound A (200 mg, 1.04 mmol, 1 eq) in dry THF(8 mL) was added lithium aluminium hydride (1 M in THF, 2.1 mL, 2.09mmol, 2 eq) drop wise at 0° C. and the resulting mixture was stirred for1 h at 0° C. The reaction mixture was quenched with solid sodium sulfatedecahydrate and filtered through Celite. The organic components wereextracted with ethyl acetate (100 mL) and the organic layer wasconcentrated in vacuo to obtain the compound B (80 mg, 39%) as colorlesssticky material which was used in the next step without furtherpurification.

¹H NMR (400 MHz, DMSO-d₆) δ 7.33 (d, J=8 Hz, 2H), 7.09 (d, J=8 Hz, 2H),2.73 (s, 2H), 2.18-2.11 (m, 4H), 2.00-1.93 (m, 1H), 1.78-1.74 (m, 1H);

LCMS: m/z=196.2 [M+H], RT=2.53 minutes; (Program R1, Column Y).

Step 1: Preparation of C-quinolin-3-yl-methylamine (B)

To a stirred solution of compound A (250 mg, 1.58 mmol, 1 eq) in MeOH(15 mL) was added Pd/C (10% Pd, 10 mg) and the resulting mixture wasstirred for 18 h at 23° C. under hydrogen atmosphere. The reactionmixture was filtered through Celite and the filtrate was concentrated invacuo. The crude residue was purified by flash chromatography(Combiflash) using 100-200 mesh silica gel eluting with 30%methanol/dichloromethane to obtain the compound B (75 mg, 29%) ascolorless sticky material.

¹H NMR (400 MHz, DMSO-d₆) δ 8.88 (d, J=2 Hz, 1H), 8.22 (s, 1H), 7.99 (d,J=8 Hz, 1H), 7.93 (d, J=8 Hz, 1H), 7.71-7.68 (m, 1H), 7.58 (t, J=8 Hz,1H), 3.94 (s, 2H);

LCMS: m/z=158.8 [M+H], RT=1.22 minutes; (Program P1, Column W).

Step-1: Preparation of 4,5-difluoro-N-methoxy-N-methyl-2-nitro-benzamide

To the stirred solution of 4,5-difluoro-2-nitro benzoic acid (10 g, 49.2mmol, 1 eq) in CH₂Cl₂ (30 mL) were added oxalyl chloride (6.4 mL, 73.8mmol, 1.5 eq) at 0° C. and catalytic amount of DMF (0.5 mL) and theresulting solution was allowed to stir at 23° C. for 3 h. The reactionmixture was concentrated in vacuo under argon and the crude residue wasdissolved in CH₂Cl₂ (50 mL). The solution was cooled to 0° C. andWeinreb amine salt (5.3 g, 54.1 mmol, 1.1 eq) and TEA (48 ml, 344.4mmol, 7 eq) were added sequentially. The resulting mixture was stirredat 23° C. for 16 h. The reaction mixture was diluted in EtOAc (200 mL×3)and the organic layer was washed with saturated aq. NaHCO₃ solution,water and brine successively. Combined organic layer was dried overanhydrous Na₂SO₄, filtered and concentrated in vacuo. The crude materialwas purified by flash chromatography (230-400 silica gel) to obtaincompound B (7.2 g, 59%) as a sticky material.

¹H NMR (DMSO-d₆) δ: 8.47-8.42 (dd, J=12 Hz, J=8 Hz, 1H), 7.99-7.95 (dd,J=12 Hz, J=8 Hz, 1H), 3.38 (s, 3H), 3.25 (s, 3H).

LCMS: m/z=247 [M+H], RT=3.06 minutes; (Program P1, Column y).

Step-2: Preparation of 4, 5-difluoro-2-nitro-benzaldehyde

To a stirred solution of compound B (7.2 g, 29.2 mmol, 1 eq) in THF (60ml) was added DIBAL (42 ml, 73.1 mol, 2.5 eq) at −78° C. and theresulting mixture was stirred at −20° C. for 4 h. The reaction mixturewas quenched with saturated aq. NH₄Cl solution (20 ml) and diluted withEtOAc (450 ml) and water (250 ml). The organic layer was separated,washed with brine, dried over anhydrous Na₂SO₄ and concentrated invacuo. The crude material was purified by flash chromatography (230-400silica gel) to obtain compound C (5 g, 90%) as yellow solid.

¹H NMR (DMSO-d₆) δ: 10.17 (s, 1H), 8.49-8.44 (dd, J=12 Hz, J=8 Hz, 1H),8.03-7.98 (dd, J=12 Hz, J=8 Hz, 1H);

LCMS: m/z=186 [M−H], RT=3.21 minutes; (Program P1, Column y).

Step-3: Preparation of 6, 7-difluoro-quinoline-3-carboxylic Acid EthylEster

To a stirred solution of compound C (5 g, 26.7 mmol, 1 eq) in ethanol(120 ml) were added 3,3-diethoxy-propionic acid ethyl ester (13.1 ml,66.8 mmol, 2.5 eq), SnCl₂.2H₂O (54 g, 240.5 mmol, 9 eq) at 23° C. andthe resulting mixture was refluxed for 16 h. The reaction mixture wasconcentrated in vacuo, the residue was diluted with water (250 ml) andthe pH was adjusted to ˜7 with saturated aq. NaHCO₃ solution. Theorganic components were extracted with EtOAc (200 ml×2), combined ethylacetate layer was washed with water, brine, dried over anhydrous Na₂SO₄,concentrated in vacuo. The crude material which was purified by flashchromatography (230-400 silica gel) with 25% EtOAc/hexane to obtaincompound D (3.8 g, 60%) as light yellow solid.

¹H NMR (DMSO-d₆) δ: 9.29 (s, 1H), 9.00 (s, 1H), 8.34-8.29 (dd, J=12 Hz,J=8 Hz, 1H), 8.16-8.11 (dd, J=12 Hz, J=8 Hz, 1H), 4.41 (q, J=8 Hz, 1H),1.38 (t, 3H).

LCMS: m/z=237.8 [M+H], RT=3.43 minutes; (Program P1, Column v).

Step-4: Preparation of 6, 7-difluoro-quinoline-3-carboxylic Acid

To a stirred solution of compound D (3.2 g, 13.5 mmol, 1 eq) in THF (10ml) was added a solution of LiOH (1.7 gm) in water (10 ml) and theresulting mixture was stirred at 23° C. for 5 h. The reaction mixturewas diluted with water (200 ml) and the pH was adjusted to 6 withsaturated aq. citric acid solution. The organic components wereextracted with EtOAc (300 ml) and ethyl acetate layer was washed withwater and brine. Ethyl acetate layer was dried over anhydrous Na₂SO₄,filtered and concentrated in vacuo to obtain compound E (2.76 g, 95%) aswhite solid which was used in the next step without furtherpurification.

¹H NMR (DMSO-d₆) δ: 13.60 (br s, 1H), 9.3 (s, 1H), 8.99 (s, 1H),8.32-8.27 (dd, J=12 Hz, J=8 Hz, 1H), 8.15-8.10 (dd, J=12 Hz, J=8 Hz,1H);

LCMS: m/z=210.2 [M+H], RT=1.83 minutes; (Program P1, Column y).

Step-5: Preparation of 7-fluoro-quinoline-3-carboxylic Acidmethoxy-methyl-amide

To a stirred solution of compound E (2.9 g, 13.8 mmol, 1 eq) in DMF (20ml) were added EDCI.HCl (3.17 g, 16.5 mmol, 1.19 eq), HOBT (2.23 g, 16.5mmol, 1.19 eq), and TEA (6 ml, 41.4 mmol, 3 eq) at 0° C. and the mixturewas stirred for 10 minutes. Weinreb amine salt (1.48 g, 15.1 mmol, 1.09eq) was added to it and the resulting mixture was stirred at 23° C. for16 h. The reaction mixture was diluted with EtOAc (350 ml) and theorganic layer was washed with saturated aq. NaHCO₃ solution (150 ml),water and brine. The organic layer was dried over anhydrous Na₂SO₄,filtered and concentrated in vacuo. The crude material was purified byCombiflash column chromatography, eluting with 25% EtOAc/hexane toobtain compound F (2 g, 57%) as white solid.

¹H NMR (DMSO-d₆) δ: 9.07 (s, 1H), 8.69 (s, 1H), 8.25-8.20 (dd, J=12 Hz,J=8 Hz, 1H), 8.13-8.08 (dd, J=12 Hz, J=8 Hz, 1H), 3.57 (s, 3H), 3.34 (s,3H);

LCMS: m/z=253 [M+H], RT=2.82 minutes; (Program P1, Column v).

Step-6: Preparation of 1-(6,7-difluoro-quinolin-3-yl)-ethanone

To a stirred solution of compound F (0.9 g, 3.57 mmol, 1 eq) in dry THF(12 ml) was added MeLi (2.5 ml, 3.93 mmol, 1.1 eq) at −78° C. and theresulting mixture was stirred at −20° C. for 3 h. The reaction mixturewas quenched with saturated aq. KHSO₄ solution (5 ml) at −20° C. anddiluted with EtOAc (150 ml). The organic layer was washed with saturatedaq. NaHCO₃ solution (100 ml), water and brine. The organic layer wasdried over anhydrous Na₂SO₄, filtered and concentrated in vacuo. Thecrude material was purified by Combiflash column chromatography elutingwith 19-25% EtOAc/hexane to obtain compound G (0.35 g, 47%) as anoff-white solid.

¹H NMR (DMSO-d₆) δ: 9.34-9.33 (d, J=4 Hz, 1H), 9.04-9.03 (d, J=4 Hz,1H), 8.29-8.24 (dd, J=12 Hz, J=8 Hz, 1H), 8.17-8.12 (dd, J=12 Hz, J=8Hz, 1H), 2.72 (s, 1H);

LCMS: m/z=208 [M+H], RT=2.91 minutes; (Program P1, Column v).

Step-7: Preparation of 1-(6, 7-difluoro-quinolin-3-yl)-ethylamine

To a stirred solution of compound G (0.7 g, 3.38 mmol, 1 eq) inmethanolic ammonia (10 ml) was added titanium isopropoxide (2 ml, 6.8mmol, and 2 eq) at 0° C. and the mixture was stirred at 23° C. for 16 h.NaBH₄ (192 mg, 5.07 mmol, 1.5 eq) was added to it at 0° C. and theresulting mixture was stirred at 23° C. for 16 h. The reaction mixturewas quenched with ice-cold water (5 ml) and concentrated in vacuo. Thesolid residue was diluted with 10% MeOH/CH₂Cl₂ (25 ml) and the mixturewas stirred for 30 min at 23° C. The mixture was filtered and thefiltrate was concentrated in vacuo. The crude material was purified byflash chromatography on neutral alumina column eluting with 7-10%MeOH/CH₂Cl₂ to obtain compound H (0.5 g, 71%) as a sticky solid.

¹H NMR (DMSO-d₆) δ: 8.95 (s, 1H), 8.28 (s, 1H), 8.05-7.96 (m, 2H), 4.21(q, J=8 Hz, 1H), 2.06 (br s, 2H), 1.36-1.34 (d, J=8 Hz, 3H);

LCMS: m/z=208.8 [M+H], RT=2.16 minutes; (Program P1, Column v).

Step-1: Preparation of pyrazolo[1,5-a]pyridine-2-carboxylic Acidmethoxy-methyl-amide

To a stirred solution of compound A (500 mg, 3.08 mmol, 1 eq) in CH₂Cl₂(15 ml) was added thionyl chloride (0.665 ml, 9.25 mmol, 3 eq) and themixture was refluxed for 3 h. The mixture was cooled to RT, concentratedin vacuo and the residue was dissolved in CH₂Cl₂ (20 ml). TEA (3 ml,21.51 mmol, 7 eq) followed by Weinreb amine salt (450 mg, 4.62 mmol, and1.5 eq) was added to it and the resulting mixture was stirred at 23° C.for 16 h. The reaction mixture was diluted with ethyl acetate (150 ml),the combined organic layer was washed with water and brine. The organiclayer was dried over anhydrous sodium sulfate and concentrated in vacuo.The crude material was purified by column chromatography to obtaincompound B (400 mg, 63%) as liquid compound.

¹H NMR (DMSO-d₆) δ 8.72-8.70 (d, J=8 Hz, 1H), 7.76-7.74 (d, J=9 Hz, 1H),7.30-7.26 (m, 1H), 7.02-7.00 (t, J=7 Hz, 1H), 3.73 (s, 3H), 3.37 (s,3H);

LCMS: m/z=206.0 [M+H], RT=2.27 minutes; (Program P1, Column v.

Step-2: Preparation of 1-pyrazolo[1,5-a]pyridin-2-yl-ethanone

To a solution of compound B (1 g, 4.88 mmol, 1 eq) in THF (20 ml) wasadded MeLi (3.5 ml, 5.36 mmol, 1.1 eq) at −78° C. and the resultingmixture was stirred for 2 h at the same temperature. The reactionmixture was quenched with satd. aq. ammonium chloride solution (10 ml)and the organic components were extracted with ethyl acetate (200 ml).Combined organic layer was washed with water and brine, dried oversodium sulfate and concentrated in vacuo. The crude material waspurified by column chromatography (eluting with 30% ethylacetate/hexane) to obtain compound C (300 mg, 38%) as liquid.

¹H NMR (DMSO-d₆) δ 8.79-8.77 (d, J=7 Hz, 1H), 7.80-7.78 (d, J=9 Hz, 1H),7.32-7.28 (t, J=7 Hz, 1H), 7.09-7.07 (d, J=7 Hz, 1H), 2.65-2.62 (s, 3H);

LCMS: m/z=206.0 [M⁺H], RT=2.27 minutes; (Program P1, Column v).

Step-3: Preparation of 1-pyrazolo[1,5-a]pyridin-2-yl-ethylamine

To a solution of compound C (270 mg, 1.69 mmol, 1 eq) in methanol (15ml) were added ammonium acetate (1.3 g, 16.87 mmol, 10 eq), sodiumcyanoborohydride (74 mg, 1.18 mmol, 0.7 eq) and molecular sieves (3 Å)and the resulting mixture was stirred at RT for 16 h. The reactionmixture was filtered and the filtrate was concentrated in vacuo. Theresidue was diluted with CH₂Cl₂, and extracted with 1N aq. HCl solutionuntil acidic pH. The aqueous layer was basified with 5N aq. NaOHsolution, and the organic components were extracted with 15%methanol/chloroform. The organic layer was concentrated in vacuo toobtain compound D (120 mg, 44%) as liquid.

¹H NMR (DMSO-d₆) δ 8.66-8.65 (m, 4H), 7.72-7.70 (d, J=9 Hz, 1H),7.27-7.21 (m, 1H), 6.94-6.92 (t, J=8 Hz, 1H), 6.74 (s, 1H), 4.62-4.56(m, 1H), 1.62-1.60 (d, J=7 Hz, 3H);

LCMS: m/z=162.2 [M⁺H], RT=1.27 minutes; (Program P1, Column y).

Step-1: Preparation of 1-(4-pyridin-2-yl-phenyl)-ethanone

To a stirred solution of compound A (0.58 g, 3.87 mmol, 1 mmol) in IPA(10 ml) was added 4-acetyl-phenyl-boronic acid (0.8 g, 4.87 mmol, 1.25eq) and the mixture was stirred at 23° C. for 15 minutes. Pd(OAc)₂ (45mg, 0.2 mmol, 0.05 eq), PPh₃ (11 mg, 0.04 mmol, 0.01 eq), 2M aqueousNa₂CO₃ (3.2 ml, 6.4 mmol, 1.65 eq) and distilled water (2 ml) were addedto it and the resulting mixture was refluxed for 16 h under nitrogenatmosphere. The reaction mixture was cooled to RT, diluted with waterand the organic components were extracted with ethyl acetate. Theorganic layer was washed with aq. Na₂CO₃ solution (0.5M, 100 ml), brine,dried over anhyd. sodium sulphate and concentrated in vacuo. The crudematerial was purified by Combiflash eluting with 15%-20% EtOAc/hexane toobtain compound B (0.6 g, 75%).

¹H NMR (DMSO-d₆) δ 8.73-8.72 (m, 1H), 8.25-8.23 (m, 2H), 8.08-8.06 (m,3H), 7.96-7.92 (m, 1H), 7.44-7.41 (m, 1H), 2.63 (s, 3H);

LCMS: m/z=198 [M+H], RT=3.01 min minutes; (Program P1, Column y).

Step-2: Preparation of 1-(4-Pyridin-2-yl-phenyl)-ethylamine

To a stirred solution of compound B (0.2 g, 1.01 mmol, 1 eq) in EtOH (2ml) were added NaCNBH₃ (76 mg, 1.21 mmol, 1.2 eq) and NH₄OAc (1.16 g,15.15 mmol, 15 eq) and the resulting mixture was heated under microwaveat 130° C. for 2 min. The reaction mixture was cooled to RT andconcentrated in vacuo. The residue was diluted with water and the aq.Layer was washed with ethyl acetate. The aqueous layer was basified withaq. NaOH solution (2N, 50 ml) and the organic components were extractedwith ethyl acetate. The combined organic layer was dried over anhydroussodium sulfate and concentrated in vacuo. The crude material waspurified by column chromatography (neutral alumina) eluting with 1-2%methanol/CH₂Cl₂ to obtain compound C (0.09 g, 56%) as solid.

¹H NMR (DMSO-d₆) δ 8.65-8.64 (d, J=4 Hz, 1H), 8.02-8.00 (d, J=8 Hz, 2H),7.93-7.91 (m, 1H), 7.86-7.83 (m, 1H), 7.48-7.49 (d, J=8 Hz, 2H),7.33-7.30 (m, 1H), 4.06-4.01 (m, 1H), 1.88 (s, 2H), 1.35-1.31 (s, 3H);

LCMS: m/z=199.1 [M+H], RT=1.66 minutes; (Program P1, Column v).

Step 1: Preparation of 1-quinolin-3-yl-ethanol (B)

To a stirred solution of compound A (20 g, 127.4 mmol, 1 eq) in dry THF(200 mL) was added methyl magnesium bromide (3 M in diethyl ether, 85mL, 254.8 mmol, 2 eq) drop wise at −78° C. and the resulting mixture wasstirred for 18 h at 23° C. The reaction mixture was quenched withsaturated aq. NH₄Cl solution and the organic components were extractedwith ethyl acetate (800 ml). The organic layer was concentrated in vacuoto obtain the compound B (20.7 g) as colorless sticky material which wasused in the next step without further purification.

¹H NMR (400 MHz, DMSO-d₆) δ 8.91 (m, 1H), 8.24 (s, 1H), 7.98 (t, J=8 Hz,2H), 7.71 (t, J=8 Hz, 1H), 7.588 (t, J=8 Hz, 1H), 5.47 (d, J=4 Hz, 1H),5.00-4.94 (m, 1H), 1.46 (d, J=7 Hz, 3H);

LCMS: m/z=174.4 [M+H], RT=1.24 minutes; (Program R1, Column Y).

Step 2: Preparation of 1-quinolin-3-yl-ethanol (C)

To a stirred solution of compound B (19.7 g, 113.9 mmol, 1 eq) in dryCH₂Cl₂ (200 mL) was added triethyl amine (24 mL, 170.8 mmol, 1.5 eq) andmethane sulphonyl chloride (13.5 mL, 170.8 mmol, 1.5 eq) drop wise at 0°C. and the resulting mixture was stirred for 14 h at 23° C. The reactionmixture was quenched with water and the organic components wereextracted with CH₂Cl₂ (500 ml). DCM layer was concentrated in vacuo toobtain the compound C (28 g) as brownish sticky material which was usedin the next step without further purification.

¹H NMR (400 MHz, DMSO-d₆) δ 9.47 (m, 1H), 9.30 (s, 1H), 8.40-8.35 (m,2H), 8.13 (t, J=8 Hz, 1H); 7.95 (t, J=8 Hz, 1H), 5.79-5.74 (m, 1H), 2.44(s, 3H), 1.98 (d, J=8 Hz, 3H);

Step 3: Preparation of 3-(1-azido-ethyl)-quinoline (D)

To a stirred solution of compound C (28 g, 111.5 mmol, 1 eq) in dry DMF(150 mL) was added sodium azide (73 g, 1.11 mol, 10 eq) in smallportions at 0° C. and the resulting mixture was stirred for 14 h at 23°C. The reaction mixture was quenched with ice and the organic componentswere extracted with ethyl acetate (500 ml). The organic layer wasconcentrated in vacuo to obtain the compound D (32 g) as brownish stickymaterial which was used in the next step without further purification.

Step 4: Preparation of 1-quinolin-3-yl-ethylamine (E)

To a stirred solution of compound D (20 g, 100 mmol, 1 eq) in a mixtureof THF (300 mL) and H₂O (30 ml) was added triphenylphosphine (40 g, 150mmol, 1.5 eq) and the resulting mixture was stirred at 70° C. for 18 h.The reaction mixture was cooled to RT, and concentrated in vacuo. Thecrude material was purified by flash chromatography (Combiflash) using100-200 mesh silica gel eluting with 10% methanol/dichloromethane toobtain the compound E (15 g, 86%) as brownish sticky material.

¹H NMR (400 MHz, DMSO-d₆) δ 8.94 (m, 1H), 8.24 (m, 1H), 7.98 (d, J=8 Hz,1H), 7.93 (d, J=8 Hz, 1H), 7.71-7.67 (m, 1H), 7.59-7.55 (m, 1H),4.25-4.20 (m, 1H), 2.16 (br s, 2H), 1.37 (d, J=8 Hz, 3H);

LCMS: m/z=173.0 [M+H], RT=1.74 minutes; (Program P1, Column Y).

Step 1: Preparation of cyclopropyl-quinolin-3-yl-methanol (B)

To a stirred solution of compound A (8 g, 0.05 mol, 1 eq) in dry THF(200 mL) was added cyclopropyl magnesium bromide (0.5 M in THF, 203 mL,0.10 mol, 2 eq) drop wise at −40° C. and the resulting mixture wasstirred for 18 h at 23° C. The reaction mixture was quenched withsaturated aq. NH₄Cl solution and the organic components were extractedwith ethyl acetate (500 ml). The organic layer was concentrated in vacuoto obtain the compound B (10 g, 80%) as colorless sticky material whichwas used in the next step without further purification.

¹H NMR (400 MHz, DMSO-d₆) δ 8.95 (s, 1H), 8.27 (s, 1H), 8.01-7.97 (m,2H), 7.74-7.70 (m, 1H), 7.61-7.57 (m, 1H), 5.50 (s, 1H), 4.25-4.22 (m,1H), 1.23-1.12 (m, 1H), 0.56-0.46 (m, 4H);

LCMS: m/z=199.8 [M+H], RT=2.75 minutes; (Program P1, Column Y).

Step 2: Preparation of cyclopropyl-quinolin-3-yl-methanone (C)

To a stirred solution of compound B (8 g, 0.05 mol, 1 eq) in CH₂Cl₂ (100mL) was added Dess-Martin Periodinane at 0° C. and the resulting mixturewas stirred at 23° C. for 16 h. The reaction mixture was filteredthrough Celite, filtrate was neutralized with aq. NaHCO₃ solution andthe organic parts were extracted with CH₂Cl₂ (500 ml). DCM layer wasconcentrated in vacuo and the crude residue was purified by flashchromatography (Combiflash) using 100-200 mesh silica gel eluting with20% ethyl acetate/hexane to obtain the compound C (7 g, 71%) as offwhite solid.

¹H NMR (400 MHz, DMSO-d₆) δ 9.38 (s, 1H), 9.19 (s, 1H), 8.20 (d, J=8 Hz,1H), 8.11 (d, J=8 Hz, 1H), 1.95-1.91 (m, 1H), 7.76-7.72 (m, 1H),3.14-3.07 (m, 1H), 1.21-1.14 (m, 4H);

LCMS: m/z=198.4 [M+H], RT=3.68 minutes; (Program R1, Column W).

Step 3: Preparation of C-cyclopropyl-C-quinolin-3-yl-methylamine (D)

To a stirred solution of compound C (4.5 g, 20 mmol, 1 eq) in methanolicammonia (7 N, 100 mL) was added titanium isopropoxide (13.5 mL, 50 mmol,2.5 eq) drop wise at 0° C. and the mixture was stirred for 24 h at 23°C. Sodium borohydride (1.3 g, 30 mmol, 1.5 eq) was added to it in smallportions at 0° C. and the resulting mixture was stirred for 18 h at 23°C. The reaction mixture was quenched with ice and concentrated in vacuo.The solid was diluted with 10% MeOH/CH₂Cl₂, the mixture was stirred for20 min., filtered, the filtrate was concentrated in vacuo. The cruderesidue was purified by flash chromatography using neutral aluminaeluting with 6% methanol/dichloromethane to obtain the compound D (2.8g, 71%) as brownish sticky material.

¹H NMR (400 MHz, DMSO-d₆) δ 8.98 (s, 1H), 8.28 (br s, 1H), 8.00-7.94 (m,2H), 7.72-7.68 (m, 1H), 7.60-7.56 (m, 1H), 3.41 (d, J=8 Hz, 1H), 2.20(br s, 2H), 1.23-1.04 (m, 1H), 0.55-0.49 (m, 4H);

LCMS: m/z=198.8 [M+H], RT=1.88 minutes; (Program P1, Column W).

Synthesis of Other Amine Intermediates:

The amines (I-V) were synthesized with a combination of above-mentionedprotocols, using commercially available appropriate precursors, as wouldbe apparent to one of ordinary skill in the art.

Synthesis Schemes for Compounds of Formula (I):

The compounds of formula (I) can be synthesized according to the schemesshown in the Examples below. It will be apparent and understood by oneof ordinary skill in the art that the chemical structures representedbelow that contain heteroatoms may, in certain circumstances, have oneor more bound hydrogens that are not shown.

Example 1

The following Scheme 19 is a representative synthesisof—1-{4-[2-Isopropyl-7-((R)-1-quinolin-3-yl-ethylamino)-2H-pyrazolo[4,3-d]pyrimidin-5-yl]-piperazin-1-yl}-ethanone):

4-Nitro-1H-pyrazole-3-carboxylic Acid Methyl Ester (II)

To a stirred solution of compound I (10 g, 64 mmol, 1 eq) in methanol(100 ml) was added SOCl₂ (5.2 ml, 67 mmol, 1.05 eq) drop wise atice-cold condition and the resulting mixture was stirred at 23° C. for16 h. The reaction mixture was concentrated in vacuo, the residue wasdiluted with water (25 ml) and the organic components were extractedwith ethyl acetate (200 ml). The ethyl acetate layer was removed invacuo to obtain the desired compound II (10.5 g, 96%) as off white solidwhich was used in the next step without further purification.

¹H NMR (400 MHz, DMSO-d₆) δ 14.41 (br s, 1H), 8.97 (s, 1H), 3.88 (s,3H);

LCMS: m/z=172.0 [M+H], RT=2.12 minutes; (Program Q1, Column X).

1-Isopropyl-4-nitro-1H-pyrazole-3-carboxylic Acid Methyl Ester (III)

To a solution of compound II (2 g, 11 mmol, 1 eq) in DMF (10 mL) wereadded K₂CO₃ (2 g, 14 mmol, 1.3 eq) and 2-bromo propane (2.06 mL, 22mmol, 2 eq) drop wise at 0° C. and the resulting mixture was stirred for18 h at 23° C. The reaction mixture was diluted with water (50 ml), theorganic components were extracted with ethyl acetate (100 ml), ethylacetate layer was concentrated in vacuo. The crude residue was purifiedby flash chromatography (Combiflash) using 100-200 mesh silica gel andeluting with 24% ethylacetate/hexane to obtain compound III (1.7 g, 72%)as pale yellow sticky material

¹H NMR (400 MHz, DMSO-d₆) δ 8.16 (s, 1H), 4.58-4.55 (m, 1H), 3.98 (s,3H), 1.57-1.52 (m, 6_H);

LCMS: m/z=214.0 [M+H], RT=3.03 minutes; (Program Q1, Column X).

4-Amino-1-isopropyl-1H-pyrazole-3-carboxylic Acid Methyl Ester (IV)

To a solution of compound III (200 mg, 0.94 mol, 1 eq) in methanol (10mL) was added Pd/C (10% Pd, 10 mole %) and the resulting mixture wasstirred at 23° C. under hydrogen atmosphere for 14 h. The reactionmixture was filtered through Celite and the filtrate was concentrated invacuo to get compound IV (150 mg, 86%) as brown solid which was used inthe next step without further purification.

¹H NMR (400 MHz, DMSO-d₆) δ 7.18 (s, 1H), 4.65 (br s, 2H), 4.42-4.36 (m,1H), 3.74 (s, 3H), 1.39-1.35 (m, 6H);

LCMS: m/z=184.2 [M+H], RT=1.20 minutes; (Program Q1, Column Y).

2-Isopropyl-2,4-dihydro-pyrazolo[4,3-d]pyrimidine-5,7-dione (V)

To compound IV (700 mg, 3.78 mol, 1 eq) was added urea (1.14 g, 18.9mmol, 5 eq) and the mixture was heated in a sealed tube at 160° C. for16 h. The reaction mixture cooled to RT and diluted with water (50 mL),filtered to obtain compound V (600 mg, 76%) as an off-white solid whichwas used in the next step without further purification.

¹H NMR (400 MHz, DMSO-d₆) δ 10.84 (s, 1H), 10.74 (s, 1H), 7.68 (s, 1H),4.65-4.58 (m, 1H), 1.43 (d, J=7 Hz, 6H).

5,7-Dichloro-2-isopropyl-2H-pyrazolo[4,3-d]pyrimidine (VI)

To compound V (500 mg, 2.58 mmol, 1 eq) were added POCl₃ (20 mL) anddiethyl aniline (1.1 mL, 6.70 mmol, 2.6 eq) drop wise under ice-coldcondition and the resulting mixture was stirred for 10 h at 80° C. Thereaction mixture was cooled to RT, concentrated in vacuo and the residuewas quenched with ice. pH of the solution was adjusted to ˜7 by aq.ammonia (3-4 mL) and the organic components were extracted with ethylacetate (100 ml) and ethyl acetate layer was concentrated in vacuo. Thecrude material was purified by flash chromatography (Combiflash) using100-200 mesh silica gel eluting with 3% ethyl acetate/hexane to obtaincompound VI (250 mg, 42%) as yellow solid.

¹H NMR (400 MHz, DMSO-d₆) δ 8.17 (s, 1H), 4.94-4.87 (m, 1H), 1.69 (d,J=7 Hz, 6H);

LCMS: m/z=231.2 [M+H], RT=3.19 minutes; (Program R1, Column Y).

(5-Chloro-2-isopropyl-2H-pyrazolo[4,3-d]pyrimidin-7-yl)-((R)-1-quinolin-3-ylethyl)Amine (VII)

To a solution of compound VI (60 mg, 0.26 mmol, 1 eq) in tert-butylalcohol (3 mL) were added (R)-1-quinolin-3-yl-ethylamine (54 mg, 0.31mmol, 1.2 eq) and DIPEA (0.10 mL, 0.52 mmol, 2 eq) and the resultingmixture was stirred at 23° C. for 24 h. The reaction mixture wasconcentrated in vacuo and the residue was diluted with water (10 mL).The organic components were extracted with CH₂Cl₂ (30 mL), DCM layer wasdried over anhydrous sodium sulfate and concentrated in vacuo. The crudematerial was purified by flash chromatography (Combiflash) using 100-200mesh silica gel eluting with 65% ethyl acetate/hexane to obtain compoundVII (60 mg, 63%) as off white foamy solid.

¹H NMR (400 MHz, DMSO-d₆) δ 9.27 (d, J=8 Hz, 1H), 9.04 (d, J=2 Hz, 1H),8.40 (s, 1H), 8.33-8.32 (m, 1H), 8.00-7.96 (m, 2H), 7.75-7.70 (m, 1H),7.61-7.58 (m, 1H), 5.69-5.65 (m, 1H), 4.82-4.77 (m, 1H), 1.72-1.67 (m,3H), 1.56-1.54 (m, 6H);

LCMS: m/z=367.4 [M+H], RT=2.91 minutes; (Program R1, Column Y).

1-{4-[2-Isopropyl-7-((R)-1-quinolin-3-yl-ethylamino)-2H-pyrazolo[4,3-d]pyrimidin-5-yl]-piperazin-1-yl}-ethanone(VIII)

To a solution of VII (50 mg, 0.14 mmol, 1 eq) in n-butyl alcohol (3 mL)were added N-acetyl piperazine (35 mg, 0.27 mmol, 2 eq) and DIPEA (0.12mL, 0.68 mmol, 5 eq) and the resulting mixture was refluxed for 48 h.The reaction mixture was cooled to RT, concentrated in vacuo and theresidue was diluted with water (30 mL). The organic components wereextracted with CH₂Cl₂ (40 mL), DCM layer was dried over anhyd. sodiumsulfate and concentrated in vacuo. The crude material was finallypurified by flash chromatography (Combiflash) using 100-200 mesh silicagel eluting with 4% MeOH/CH₂Cl₂ to obtain compound VIII (50 mg, 78%) asoff white solid.

¹H NMR (400 MHz, DMSO-d₆) δ 9.05 (d, J=2 Hz, 1H), 8.49 (d, J=7 Hz, 1H),8.33-8.33 (m, 1H), 7.96 (t, J=8 Hz, 2H), 7.91 (s, 1H), 7.71-7.67 (m,1H), 7.59-7.56 (m, 1H), 5.53 (t, J=7 Hz, 1H), 4.69-4.66 (m, 1H),3.59-3.25 (m, 8H), 1.97 (s, 3H), 1.69 (d, J=8 Hz, 3H), 1.68-1.52 (m,6H);

LCMS: m/z=459.4 [M+H], RT=2.29 minutes; (Program R1, Column Y).

Examples 2-8

The 7 compounds of formula (I) identified in Table 1 were synthesizedusing Scheme 19.

TABLE 1 Example No. Molecular Structure LCMS 2

LCMS: m/z = 459.4 [M + H], RT = 2.09 minutes; (Program R1, Column X). 3

LCMS: m/z = 470.4 [M + H], RT = 2.90 minutes; (Program R1, Column Y). 4

LCMS: m/z = 473.4 [M + H], RT = 2.50 minutes; (Program R1, Column Y). 5

LCMS: m/z = 473.0 [M + H], RT = 2.50 minutes; (Program R1, Column Y). 6

LCMS: m/z = 483.8 [M + H], RT = 2.99 minutes; (Program R1, Column Y). 7

LCMS: m/z = 472.8 [M + H], RT = 2.48 minutes; (Program R1, Column Y). 8

LCMS: m/z = 487.4 [M + H], RT = 2.67 minutes; (Program R1, Column Y).

Example 9—Scheme 20: Synthesis of1-{4-[2-Phenyl-7-((R)-1-quinolin-3-yl-ethylamino)-2H-pyrazolo[4,3-d]pyrimidin-5-yl]-piperazin-1-yl}ethanone

4-Nitro-1-phenyl-1H-pyrazole-3-carboxylic Acid Methyl Ester (IIIa)

To a solution of compound II (200 mg, 1.17 mmol, 1 eq) in dry CH₂Cl₂ (15mL) were added phenyl boronic acid (285 mg, 2.34 mmol, 2 eq), pyridine(0.2 mL, 2.34 mmol, 2 eq) and Cu(OAc)₂.H₂O (350 mg, 1.75 mmol, 1.5 eq)and the resulting mixture was refluxed for 14 h. The reaction mixturewas filtered through Celite, solids were washed with CH₂Cl₂ (100 ml) andthe combined filtrate was concentrated in vacuo. The crude residue waspurified by flash chromatography (Combiflash) using 100-200 mesh silicagel eluting with 24% ethyl acetate/hexane to obtain compound IIIa (230g, 80%) as colorless sticky material.

¹H NMR (400 MHz, DMSO-d₆) δ 9.73 (s, 1H), 7.94 (d, J=8 Hz, 2H),7.62-7.58 (m, 2H), 7.52-7.50 (m, 1H), 3.95 (s, 3H);

LCMS: m/z=248.0 [M+H], RT=3.32 minutes; (Program R1, Column Y).

Steps 3 to 7 were carried out according to the process described inScheme 19 to obtain the compound of Example 9 (shown in Table 2 below)as an off white solid.

TABLE 2 Example 9

LCMS: m/z = 493.0 [M + H], RT = 2.61 minutes; (Program R1, Column Y).

Example 10—Scheme 21 Below is a Representative Synthesisof—4-{1-sec-Butyl-7-[((R)-cyclopropyl-quinolin-3-yl-methyl)-amino]-1H-pyrazolo[4,3-d]pyrimidin-5-yl}-piperazine-1-carboxylicAcid Amide)

4-Nitro-1H-pyrazole-3-carboxylic Acid Methyl Ester (II)

To a stirred solution of compound I (10 g, 64 mmol, 1 eq) in methanol(100 ml) was added SOCl₂ (5.2 ml, 67 mmol, 1.05 eq) drop wise atice-cold condition and the resulting mixture was stirred for 16 h at 23°C. The reaction mixture was concentrated in vacuo and the residue wasdiluted with water (25 ml). The organic components were extracted withethyl acetate (200 ml), ethyl acetate layer was concentrated in vacuo toobtain the desired compound II (10.5 g, 96%) as off white solid materialwhich was used in the next step without further purification.

¹H NMR (400 MHz, DMSO-d) δ 14.41 (br s, 1H), 8.97 (s, 1H), 3.88 (s, 3H);

LCMS: m/z=172.0 [M+H], RT=2.12 minutes; (Program Q1, Column X).

2-sec-Butyl-4-nitro-2H-pyrazole-3-carboxylic Acid Methyl Ester (III)

To a solution of compound II (5 g, 0.03 mol, 1 eq) in dry THF (100 mL)were added butan-2-ol (4.3 g, 58 mmol, 2 eq) and triphenyl phosphine(15.3 g, 58 mmol, 2 eq) under ice-cold condition. DIAD (12 mL, 58 mmol,2 eq) was added to it drop wise at 0° C. and the resulting mixture wasstirred for 3 h at the same temperature. The reaction mixture wasconcentrated in vacuo, the residue was diluted with water (100 ml) andthe organic components were extracted with ethyl acetate (250 ml). Ethylacetate layer was concentrated in vacuo and the crude residue waspurified by flash chromatography (Combiflash) using 100-200 mesh silicagel eluting with 5% ethyl acetate/hexane to obtain compound III (4 g,60%) as pale yellow sticky material.

¹H NMR (400 MHz, DMSO-d₆) δ 8.43 (s, 1H), 4.44-4.39 (m, 1H), 3.32 (s,3H), 1.90-1.74 (m, 2H), 4.44-4.43 (m, 3H), 0.52-0.32 (m, 3H);

LCMS: m/z=228.2 [M+H], RT=5.10 minutes; (Program R2, Column W).

4-Amino-2-sec-butyl-2H-pyrazole-3-carboxylic Acid Methyl Ester (IV)

To a solution of compound III (4.5 g, 20 mmol, 1 eq) in methanol (150mL) was added Pd/C (10% Pd, 10 mole %) and the resulting mixture wasstirred at 23° C. under hydrogen atmosphere for 14 h. The reactionmixture was filtered through Celite and the filtrate was concentrated invacuo to get compound IV (2 g, 89%) light brown sticky material whichwas used in the next step without further purification.

¹H NMR (400 MHz, DMSO-d₆) δ 7.08 (s, 1H), 5.07-5.02 (m, 1H), 4.99-4.92(m, 2H), 3.79 (s, 3H), 1.83-1.74 (m, 1H), 1.68-1.60 (m, 1H), 1.29 (d,J=7 Hz, 3H), 0.70-0.66 (m, 3H)

LCMS: m/z=198.1 [M+H], RT=2.96 minutes; (Program R1, Column W).

1-sec-Butyl-1,4-dihydro-pyrazolo[4,3-d]pyrimidine-5,7-dione (V)

To compound IV (1 g, 5 mmol, 1 eq) urea (1.5 g, 25 mmol, 5 eq) was addedand the mixture was heated in a selared tube at 160° C. for 16 h. Thereaction mixture was cooled to RT, diluted with water (60 mL) andfiltered to obtain compound V (800 mg, 77%) as off-white solid which wasused in the next step without further purification.

¹H NMR (400 MHz, DMSO-d₆) δ 11.07-11.00 (m, 2H), 7.39 (s, 1H), 5.04-4.99(m, 1H), 1.89-1.80 (m, 1H), 1.78-1.73 (m, 1H), 1.40 (d, J=7 Hz, 3H),0.68 (t, J=7 Hz, 3H);

LCMS: m/z=207.1 [M+H], RT=2.36 minutes; (Program R1, Column W).

1-sec-Butyl-5,7-dichloro-1H-pyrazolo[4,3-d]pyrimidine (VI)

To compound V (1 g, 4.81 mmol, 1 eq) were added POCl₃ (70 mL) anddiethyl aniline (2 mL, 12.5 mmol, 2.6 eq) drop wise under ice-coldcondition and the resulting mixture was stirred for 10 h at 80° C. Thereaction mixture was cooled to RT, concentrated in vacuo, and theresidue was quenched with ice. pH of the solution was adjusted to ˜7 byaq. ammonia (3-4 mL) and the organic components were extracted withethyl acetate (200 ml). Ethyl acetate layer was concentrated in vacuo,and the crude material was purified by flash chromatography (Combiflash)using 100-200 mesh silica gel eluting with 2.5% ethyl acetate/hexane toobtain compound VI (950 mg, 81%) as yellow solid.

¹H NMR (CDCl₃) δ 8.23 (s, 1H), 5.29-5.24 (m, 1H), 2.15-2.08 (m, 1H),1.94-1.87 (m, 1H), 1.59 (d, J=7 Hz, 3H), 8.04 (t, J=7 Hz, 3H);

LCMS: m/z=245.1[M+H], RT=4.03 minutes; (Program R1, Column w).

(1-sec-Butyl-5-chloro-1H-pyrazolo[4,3-d]pyrimidin-7-yl)-((R)-cyclopropyl-quinolin-3-yl-methyl)-amine(VII)

To a solution of compound VI (190 mg, 0.78 mmol, 1 eq) in tert-butylalcohol (5 mL) were addedC—((R)—C-cyclopropyl-C-quinolin-3-yl)-methylamine (154 mg, 0.78 mmol, 1eq) and DIPEA (0.40 mL, 2.33 mmol, 3 eq) and the resulting mixture wasstirred at 23° C. for 24 h. The reaction mixture was concentrated invacuo, the residue was diluted with water (30 mL) and the organiccomponents were extracted with CH₂Cl₂ (100 mL). DCM layer was dried overanhyd. sodium sulfate and concentrated in vacuo. The crude materialwhich was purified by flash chromatography (Combiflash) using 100-200mesh silica gel eluting with 61% ethyl acetate/hexane to obtain compoundVII (200 mg, 63%) off white foamy solid.

¹H NMR (400 MHz, DMSO-d₆) δ 9.12-9.10 (m, 1H), 8.46-8.45 (m, 1H),8.22-8.19 (m, 1H), 8.07-8.06 (m, 1H), 8.00-7.96 (m, 2H), 7.76-7.72 (m,1H), 7.63-7.59 (m, 1H), 5.19-5.16 (m, 1H), 4.87-4.77 (m, 1H), 1.90-1.77(m, 3H), 1.59-1.52 (m, 3H), 0.75-0.63 (m, 7H);

LCMS: m/z=407.2 [M+H], RT=4.12 minutes; (Program R1, Column W).

4-{1-sec-Butyl-7-[((R)-cyclopropyl-quinolin-3-yl-methyl)-amino]-1H-pyrazolo[4,3-d]pyrimidin-5-yl}-piperazine-1-carboxylicAcid Tert-Butyl Ester (VIII)

To a solution of VII (200 mg, 0.49 mmol, 1 eq) in n-butyl alcohol (5 mL)were added N-BOC piperazine (183 mg, 0.99 mmol, 2 eq) and DIPEA (0.3 mL,1.48 mmol, 3 eq) and the mixture was refluxed for 48 h. The reactionmixture was cooled to RT, concentrated in vacuo, the residue was dilutedwith water (30 mL). The organic components were extracted with CH₂Cl₂(100 mL), DCM layer was dried over anhyd. sodium sulfate andconcentrated in vacuo. The crude material was purified by flashchromatography (Combiflash) using 100-200 mesh silica gel eluting with3.5% MeOH/CH₂Cl₂ to obtain compound VIII (200 mg, 73%) as light brownsolid.

¹H NMR (400 MHz, DMSO-d₆) δ 9.11-9.09 (m, 1H), 8.43-8.42 (m, 1H),8.00-7.93 (m, 2H), 7.73-7.69 (m, 2H), 7.60-7.55 (m, 2H), 5.16-4.97 (m,1H), 4.75-4.55 (m, 1H), 3.51-3.32 (m, 4H), 3.20-3.01 (m, 4H), 1.90-1.81(m, 3H), 1.58-1.50 (m, 3H), 1.39-1.38 (m, 9H), 1.25-1.23 (m, 3H),0.77-0.61 (m, 4H);

LCMS: m/z=557.5 [M+H], RT=3.44 minutes; (Program R1, Column Y).

(1-sec-Butyl-5-piperazin-1-yl-1H-pyrazolo[4,3-d]pyrimidin-7-yl)-((R)-cyclopropyl-quinolin-3-yl-methyl)-amine(IX)

To a solution of compound VIII (200 mg, 0.36 mmol, 1 eq) in CH₂Cl₂ (5mL) was added TFA (1 mL, 3.60 mmol, 10 eq) in ice-cold condition and theresulting mixture was stirred at 23° C. for 3 h. The reaction mixturewas concentrated in vacuo, the residue was diluted with toluene (3 mL)and further concentrated. The residue was then diluted with aq. NaHCO₃(3-4 mL) and the organic components were extracted with 5% MeOH/CH₂Cl₂(100 ml). Organic layer was concentrated in vacuo to obtain compound IX(160 mg, 97%) off white solid which was used in the next step withoutfurther purification.

¹H NMR (400 MHz, DMSO-d₆) δ 9.09-9.07 (m, 1H), 8.40-8.39 (m, 1H),8.00-7.97 (m, 1H), 7.95-7.91 (m, 1H), 7.73-7.69 (m, 1H), 7.67-7.66 (m,1H), 7.60-7.57 (m, 1H), 7.52-7.50 (m, 1H), 5.07-4.98 (m, 1H), 4.75-4.55(m, 1H), 3.38-3.29 (m, 8H), 1.96-1.59 (m, 3H), 1.57-1.49 (m, 3H),1.33-1.23 (m, 3H), 0.78-0.57 (m, 5H);

LCMS: m/z=457.4 [M+H], RT=2.77 minutes; (Program R1, Column Y).

4-{1-sec-Butyl-7-[((R)-cyclopropyl-quinolin-3-yl-methyl)-amino]-H-pyrazolo[4,3-d]pyrimidin-5-yl}-piperazine-1-carboxylicAcid Amide (X)

To a solution of compound IX (50 mg, 0.11 mmol, 1 eq) in THF (3 mL) wasadded TMS-isocyanate (0.03 mL, 0.22 mmol, 2 eq) at 23° C. and theresulting mixture was stirred for 14 h. The reaction mixture was dilutedwith aq. NaHCO₃ (10 mL) and the organic components were extracted withethyl acetate (70 mL). Organic layer was dried over anhydrous sodiumsulfate and concentrated in vacuo. The crude material was purified byflash chromatography (Combiflash) using 100-200 mesh silica gel elutingwith 12% MeOH/CH₂Cl₂ to obtain compound X (40 mg, 73%) as off whitesolid.

¹H NMR (400 MHz, DMSO-d₆) δ 9.11-9.08 (m, 1H), 8.42-8.40 (m, 1H),8.00-7.93 (m, 2H), 7.73-7.69 (m, 2H), 7.61-7.54 (m, 2H), 5.95-5.94 (m,2H), 5.09-5.07 (m, 1H), 4.79-4.62 (m, 1H), 4.44-3.14 (m, 8H), 1.85-1.74(m, 3H), 1.57-1.50 (m, 3H), 1.25-1.24 (m, 3H), 0.84-0.70 (m, 4H);

LCMS: m/z=500.4 [M+H], RT=2.94 minutes; (Program R1, Column Y).

Examples 11-88

The 77 compounds shown in Table 3 below were synthesized usingScheme-21.

TABLE 3 Example No. Molecular Structure LCMS 11

LCMS: m/z = 459.4 [M + H], RT = 2.32 minutes; (Program R1, Column Y). 12

LCMS: m/z = 459.4 [M + H], RT = 2.75 minutes; (Program P1, Column Y). 13

LCMS: m/z = 470.2 [M + H], RT = 2.94 minutes; (Program R1, Column Y). 14

LCMS: m/z = 445.2 [M + H], RT = 2.05 minutes; (Program R1, Column Y). 15

LCMS: m/z = 445.2 [M + H], RT = 2.32 minutes; (Program R1, Column Y). 16

LCMS: m/z = 473.0 [M + H], RT = 2.53 minutes; (Program R1, Column Y). 17

LCMS: m/z = 459.2 [M + H], RT = 2.36 minutes; (Program R1, Column Y). 18

LCMS: m/z = 473.0 [M + H], RT = 2.57 minutes; (Program R1, Column Y). 19

LCMS: m/z = 473.4 [M + H], RT = 2.62 minutes; (Program R1, Column Y). 20

LCMS: m/z = 459.4 [M + H], RT = 2.56 minutes; (Program R1, Column Y). 21

LCMS: m/z = 473.2 [M + H], RT = 2.92 minutes; (Program P1, Column W). 22

LCMS: m/z = 487.6 [M + H], RT = 2.71 minutes; (Program R1, Column Y). 23

LCMS: m/z = 459.4 [M + H], RT = 2.54 minutes; (Program R1, Column Y). 24

LCMS: m/z = 489.2 [M + H], RT = 2.48 minutes; (Program R1, Column Y). 25

LCMS: m/z = 485.2 [M + H], RT = 2.70 minutes; (Program R1, Column Y). 26

LCMS: m/z = 515.2 [M + H], RT = 2.27 minutes; (Program R1, Column Y). 27

LCMS: m/z = 501.4 [M + H], RT = 2.38 minutes; (Program R1, Column Y). 28

LCMS: m/z = 501.4 [M + H], RT = 3.08 minutes; (Program R1, Column Y). 29

LCMS: m/z = 510.0 [M + H], RT = 3.65 minutes; (Program P1, Column Y). 30

LCMS: m/z = 480.2 [M + H], RT = 3.32 minutes; (Program P1, Column Y). 31

LCMS: m/z = 496.0 [M + H], RT = 3.57 minutes; (Program P1, Column Y). 32

LCMS: m/z = 485.2 [M + H], RT = 2.72 minutes; (Program R1, Column Y). 33

LCMS: m/z = 480.2 [M + H], RT = 3.32 minutes; (Program P1, Column Y). 34

LCMS: m/z = 511.4 [M + H], RT = 2.66 minutes; (Program R1, Column Y). 35

LCMS: m/z = 500.8 [M + H], RT = 3.09 minutes; (Program R1, Column Y). 36

LCMS: m/z = 500.8 [M + H], RT = 3.10 minutes; (Program R1, Column Y). 37

LCMS: m/z = 473.0 [M + H], RT = 2.86 minutes; (Program R1, Column Y). 38

LCMS: m/z = 486.2 [M + H], RT = 3.76 minutes; (Program R1, Column Y). 39

LCMS: m/z = 475.9 [M + H], RT = 3.19 minutes; (Program R1, Column Y). 40

LCMS: m/z = 485.8 [M + H], RT = 3.35 minutes; (Program R1, Column Y). 41

LCMS: m/z = 430.7 [M + H], RT = 2.40 minutes; (Program R1, Column Y). 42

LCMS: m/z = 504.8 [M + H], RT = 3.12 minutes; (Program R1, Column Y). 43

LCMS: m/z = 486.7 [M + H], RT = 3.03 minutes; (Program R1, Column Y). 44

LCMS: m/z = 513.2 [M + H], RT = 3.08 minutes; (Program R1, Column Y). 45

LCMS: m/z = 476.5 [M + H], RT = 3.25 minutes; (Program R1, Column Y). 46

LCMS: m/z = 490.0 [M + H], RT = 3.29 minutes; (Program R1, Column Y). 47

LCMS: m/z = 494.0 [M + H], RT = 3.22 minutes; (Program R1, Column Y). 48

LCMS: m/z = 471.0 [M + H], RT = 2.79 minutes; (Program R1, Column Y). 49

LCMS: m/z = 457.4 [M + H], RT = 2.83 minutes; (Program R1, Column Y). 50

LCMS: m/z = 445.6 [M + H], RT = 2.72 minutes; (Program P1, Column Y). 51

LCMS: m/z = 488.2 [M + H], RT = 2.87 minutes; (Program R1, Column Y). 52

LCMS: m/z = 499.2 [M + H], RT = 3.15 minutes; (Program R1, Column Y). 53

LCMS: m/z = 499.1 [M + H], RT = 2.92 minutes; (Program R1, Column Y). 54

LCMS: m/z = 505.2 [M + H], RT = 3.09 minutes; (Program R1, Column Y). 55

LCMS: m/z = 516.0 [M + H], RT = 3.37 minutes; (Program R1, Column Y). 56

LCMS: m/z = 499.2 [M + H], RT = 3.02 minutes; (Program R1, Column Y). 57

LCMS: m/z = 492.2 [M + H], RT = 3.27 minutes; (Program R1, Column Y). 58

LCMS: m/z = 525.4 [M + H], RT = 3.23 minutes; (Program R1, Column Y). 59

LCMS: m/z = 471.2 [M + H], RT = 2.90 minutes; (Program R1, Column Y). 60

LCMS: m/z = 487.2 [M + H], RT = 3.00 minutes; (Program R1, Column Y). 61

LCMS: m/z = 505.2 [M + H], RT = 3.09 minutes; (Program R1, Column Y). 62

LCMS: m/z = 513.3 [M + H], RT = 3.12 minutes; (Program R1, Column Y). 63

LCMS: m/z = 533.5 [M + H], RT = 3.33 minutes; (Program R1, Column Y). 64

LCMS: m/z = 474.2 [M + H], RT = 2.30 minutes; (Program R1, Column W). 65

LCMS: m/z = 531.3 [M + H], RT = 2.86 minutes; (Program R1, Column W). 66

LCMS: m/z = 486.2 [M + H], RT = 2.31 minutes; (Program R1, Column W). 67

LCMS: m/z = 489.3 [M + H], RT = 1.72 minutes; (Program R1, Column W). 68

LCMS: m/z = 510.3 [M + H], RT = 2.71 minutes: (Program R1, Column W). 69

LCMS: m/z = 500.4 [M + H], RT = 2.57 minutes; (Program R1, Column W). 70

LCMS: m/z = 500.4 [M + H], RT = 2.57 minutes; (Program R1, Column W). 71

LCMS: m/z = 463.2 [M + H], RT = 5.25 minutes; (Program R1, Column W). 72

LCMS: m/z = 528.3 [M + H], RT = 2.25 minutes; (Program R1, Column W). 73

LCMS: m/z = 463.0 [M + H], RT = 2.86 minutes; (Program P1, Column Y). 74

LCMS: m/z = 454.6 [M + H], RT = 3.46 minutes; (Program R1, Column W). 75

LCMS: m/z = 499.3 [M + H], RT = 2.55 minutes; (Program R1, Column W). 76

LCMS: m/z = 483.4 [M + H], RT = 3.10 minutes; (Program R1, Column W). 77

LCMS: m/z = 440.4 [M + H], RT = 3.36 minutes; (Program R1, Column W). 78

LCMS: m/z = 441.4 [M + H], RT = 3.32 minutes; (Program R1, Column W). 79

LCMS: m/z = 441.4 [M + H], RT = 3.43 minutes; (Program R1, Column W). 80

LCMS: m/z = 517.0 [M + H], RT = 3.26 minutes; (Program R1, Column W). 81

LCMS: m/z = 489.3 [M + H], RT = 6.09 minutes; (Program P2, Column V). 82

LCMS: m/z = 503.2 [M + H], RT = 3.28 minutes; (Program R1, Column W). 83

LCMS: m/z = 501.4 [M + H], RT = 3.40 minutes; (Program R1, Column W). 84

LCMS: m/z = 477.4 [M + H], RT = 2.08 minutes; (Program R1, Column W). 85

LCMS: m/z = 474.2 [M + H], RT = 2.92 minutes; (Program R1, Column W). 86

LCMS: m/z = 512.2 [M + H], RT = 3.16 minutes; (Program R1, Column W). 87

LCMS: m/z = 490.2 [M + H], RT = 2.79, 2.88 minutes; (Program R1, ColumnW). 88

LCMS: m/z = 416.2 [M + H], RT = 2.93 minutes; (Program R1, Column W).

Examples 78 and 79 are isomers, and were obtained from S_(N)Ar reactionof compound VII and tetrazole. Preparative HPLC was carried out withwater/acetonitrile using 0.1% formic acid buffer to isolate the isomers.As the actual structure of a given isomer could not be determined,structures were assigned arbitrarily where Example 78 represented thepeak eluting first with shorter retention time and Example 79 elutingafterwards with higher retention time.

Example 88 was synthesized by similar S_(N)Ar strategy by refluxing withNaCN and DABCO in DMSO/water (1:1), that resulted in this compound,which is a partially hydrolyzed —CONH2 analog instead of the anticipated—CN compound.

Examples 89-93—Scheme 22 Below is a Representative Synthesis of1-{4-[1-sec-Butyl-7-((R)-1-quinolin-3-yl-ethylamino)-1H-pyrazolo[4,3-d]pyrimidin-5-yl]-piperazin-1-yl}-ethanone,1-{4-[3-Bromo-1-sec-butyl-7-((R)-1-quinolin-3-yl-ethylamino)-1H-pyrazolo[4,3-d]pyrimidin-5-yl]-piperazin-1-yl}-ethanone,5-(4-Acetyl-piperazin-1-yl)-1-sec-butyl-7-((R)-1-quinolin-3-yl-ethylamino)-1H-pyrazolo[4,3-d]pyrimidine-3-carbonitrile,1-{4-[1-sec-Butyl-3-methyl-7-((R)-1-quinolin-3-yl-ethylamino)-1H-pyrazolo[4,3-d]pyrimidin-5-yl]-piperazin-1-yl}-ethanoneand1-{4-[1-sec-Butyl-3-cyclopropyl-7-((R)-1-quinolin-3-yl-ethylamino)-1H-pyrazolo[4,3-d]pyrimidin-5-yl]-piperazin-1-yl}-ethanone

Intermediate VIIa was synthesized in a similar manner to that of VII.

Example 89:1-{4-[1-sec-Butyl-7-((R)-1-quinolin-3-yl-ethylamino)-1H-pyrazolo[4,3-d]pyrimidin-5-yl]-piperazin-1-yl}-ethanone(VIIIa)

To a solution of VIIa (100 mg, 0.26 mmol, 1 eq) in n-butyl alcohol (5mL) were added N-acetyl piperazine (41 mg, 0.32 mmol, 1.23 eq) and DIPEA(0.14 mL, 0.79 mmol, 3 eq) and the resulting mixture was refluxed for 48h. The reaction mixture was cooled to RT, concentrated in vacuo, theresidue was diluted with water (20 mL). The organic components wereextracted with CH₂Cl₂ (50 mL), DCM layer was dried over anhydrous sodiumsulfate and concentrated in vacuo. The crude material was purified byflash chromatography (Combiflash) using 100-200 mesh silica gel elutingwith 3% MeOH/CH₂Cl₂ to obtain compound VIIIa (40 mg, 31%) as off whitesolid.

¹H NMR (400 MHz, DMSO-d₆) δ 9.06-9.04 (m, 1H), 8.35 (br s, 1H),8.00-7.93 (m, 2H), 7.72-7.69 (m, 2H), 7.61-7.57 (m, 1H), 7.40-7.34 (m,1H), 5.64-5.58 (m, 1H), 5.04-5.00 (m, 1H), 3.54-3.19 (m, 8H), 1.98-1.94(m, 3H), 1.88-1.85 (m, 1H), 1.79-1.74 (m, 4H), 1.54-1.49 (m, 3H),0.75-0.66 (m, 3H);

LCMS: m/z=473.0 [M+H], RT=2.51 minutes; (Program R1, Column Y).

Example 90:1-{4-[3-Bromo-1-sec-butyl-7-((R)-1-quinolin-3-yl-ethylamino)-1H-pyrazolo[4,3-d]pyrimidin-5-yl]-piperazin-1-yl}-ethanone(VIIIb)

To a solution of compound VIIIa (30 mg, 0.06 mmol, 1 eq) in dry THF (5mL) was added NBS (12 mg, 0.06 mmol, 1 eq) at 0° C. and the resultingmixture was stirred for 20 minutes at 23° C. The reaction mixture wasdiluted with aq. NaHCO₃ (5 mL) and the organic components were extractedwith CH₂Cl₂ (20 mL), organic layer was dried over anhyd. sodium sulfateand concentrated in vacuo. The crude material was purified by flashchromatography (Combiflash) using 100-200 mesh silica gel eluting with3% MeOH/CH₂Cl₂ to obtain compound VIIIb (29 mg, 88%) as white solid.

¹H NMR (400 MHz, DMSO-d₆) δ 9.06-9.03 (m, 1H), 8.35 (s, 1H), 8.00-7.94(m, 2H), 7.73-7.69 (m, 1H), 7.61-7.53 (m, 2H), 5.63-5.61 (m, 1H),5.15-4.95 (m, 1H), 3.57-3.30 (m, 8H), 1.96 (d, J=7 Hz, 3H), 1.77-1.73(m, 5H), 1.53-1.49 (m, 3H), 0.76-0.68 (m, 3H);

LCMS: m/z=551.0 [M+], 553.0 [M+2], RT=3.09 minutes; (Program R1, ColumnY).

Example 91:5-(4-Acetyl-piperazin-1-yl)-1-sec-butyl-7-((R)-1-quinolin-3-yl-ethylamino)-1H-pyrazolo[4,3-d]pyrimidine-3-carbonitrile(VIIIc)

To a solution of compound VIIIb (25 mg, 0.05 mmol, 1 eq) in dry DMF (2mL) was added CuCN (19 mg, 0.23 mmol, 5 eq) and the resulting mixturewas heated in a sealed tube at 200° C. for 12 h. The reaction mixturewas cooled to RT, filtered through Celite and solids were washed withethyl acetate (50 mL). Combined filtrate was diluted with water (20 ml)and the organic components were extracted with ethyl acetate (20 mL).The organic layer was dried over anhyd. sodium sulfate and concentratedin vacuo. The crude material was purified by flash chromatography(Combiflash) using 100-200 mesh silica gel eluting with 3% MeOH/CH₂Cl₂to obtain compound VIIIc (5 mg 20%) as off white solid.

LCMS: m/z=498.0 [M+H], RT=3.16 minutes; (Program R1, Column Y).

Example 92:1-{4-[1-sec-Butyl-3-methyl-7-((R)-1-quinolin-3-yl-ethylamino)-1H-pyrazolo[4,3-d]pyrimidin-5-yl]-piperazin-1-yl}-ethanone(VIIId)

To a solution of compound VIIIb (40 mg, 0.07 mmol, 1 eq) in a mixture ofdioxane and water (3:2 mL) were added trimethyl boraxane (0.03 mL, 0.22mmol, 3 eq), K₂CO₃ (30 mg, 0.22 mmol, 3 eq) and the mixture was degassedwith Argon for 30 min. Pd(PPh₃)₄ was added to it and the resultingmixture was further degassed with Argon for another 15 min and heated at90° C. for 12 h. The reaction mixture was cooled to RT, filtered throughCelite and solids were washed with ethyl acetate (20 mL). The filtratewas diluted with water (20 ml) and the organic components were extractedwith ethyl acetate (50 mL), ethyl acetate layer was dried over anhydroussodium sulfate and concentrated in vacuo. The crude material waspurified by Prep TLC using 2.5% MeOH/CH₂Cl₂ to obtain compound VIIId (20mg, 59%) as off white solid.

¹H NMR (400 MHz, DMSO-d₆) δ 9.05-9.03 (m, 1H), 8.33 (s, 1H), 8.00-7.92(m, 2H), 7.70 (t, J=7 Hz, 1H), 7.60-7.58 (m, 1H), 7.34-7.28 (m, 1H),5.64-5.58 (m, 1H), 5.00-4.93 (m, 1H), 3.54-3.20 (m, 8H), 2.26 (s, 3H),1.99-1.95 (m, 3H), 1.9-1.72 (m, 5H), 1.50-1.47 (m, 3H), 0.74-0.65 (m,3H);

LCMS: m/z=487.2 [M+H], RT=2.65 minutes; (Program R1, Column Y).

Example 93:1-{4-[1-sec-Butyl-3-cyclopropyl-7-((R)-1-quinolin-3-yl-ethylamino)-1H-pyrazolo[4,3-d]pyrimidin-5-yl]-piperazin-1-yl}-ethanone

To a solution of compound VIIIb (40 mg, 0.07 mmol, 1 eq) in a mixture ofdioxane and water (3:2 mL) were added cyclopropyl boronic acid (13 mg,0.15 mmol, 2.1 eq), Na₂CO₃ (23 mg, 0.22 mmol, 3.1 eq), tricyclohexylphosphine (2 mg, 0.01 mmol, 0.1 eq) and the mixture was degassed withArgon for 15 min. Pd(OAc)₂ (1 mg, 0.004 mmol, 0.05 eq) was added to itand the resulting mixture was further degassed with Argon for another 30min and heated at 90° C. for 14 h. The reaction mixture was cooled toRT, filtered through Celite and solids were washed with ethyl acetate(20 mL). The filtrate was diluted with water and the organic componentswere extracted with ethyl acetate (50 mL), ethyl acetate layer was driedover anhydrous sodium sulfate and concentrated in vacuo. The crudematerial was purified by Prep TLC using 2.5% MeOH/CH₂Cl₂ to obtaincompound VIIIe (5 mg, 14%) as off white solid.

LCMS: m/z=513.4 [M+H], RT=3.04 minutes; (Program R1, Column Y).

Examples 94-107: The 14 compounds shown in Table 4 below weresynthesized using Scheme-22. The compound of Example 95 was obtained asa side product during the synthesis of the compound of Example 103, dueto the partial hydrolysis of the —CN group.

TABLE 4 Example No. Molecular Structure LCMS 94

LCMS: m/z = 537.2 [M+], 539.2 [M + 2], RT = 2.90 minutes; (Program R1,Column Y). 95

LCMS: m/z = 502.4 [M + H], RT = 2.72 minutes; (Program R1, Column Y). 96

LCMS: m/z = 473.4 [M + H], RT = 2.47 minutes; (Program R1, Column Y). 97

LCMS: m/z = 548.4 [M+], 550.4 [M + 2], RT = 3.66 minutes; (Program R1,Column Y). 98

LCMS: m/z = 536.8 [M+], 538.8 [M + 2], RT = 2.56 minutes; (Program R1,Column Y). 99

LCMS: m/z = 493.4 [M + H], RT = 3.07 minutes; (Program P1, Column W).100

LCMS: m/z = 536.8 [M+], 539.0 [M + 2], RT = 2.92 minutes; (Program R1,Column Y). 101

LCMS: m/z = 483.8 [M + H], RT = 2.97 minutes; (Program R1, Column Y).102

LCMS: m/z = 473.2 [M + H], RT = 2.45 minutes; (Program R1, Column Y).103

LCMS: m/z = 483.8 [M + H], RT = 2.74 minutes; (Program R1, Column Y).104

LCMS: m/z = 483.8 [M + H], RT = 2.02 minutes; (Program R1, Column Y).105

LCMS: m/z = 550.8 [M+], 552.8 [M + 2], RT = 2.73 minutes; (Program R1,Column Y). 106

LCMS: m/z = 498.2 [M + H], RT = 2.89 minutes; (Program R1, Column Y).107

LCMS: m/z = 499.2 [M + H], RT = 2.26 minutes; (Program R1, Column V).

Example 108: Scheme 23 is a Representative Synthesis of4-{1-sec-Butyl-7-[((R)-cyclopropyl-quinolin-3-yl-methyl)-amino]-1H-pyrazolo[4,3-d]pyrimidin-5-yl}-piperazine-1-sulfonicacid amide) (Xa)

To the solution of compound IX (30 mg, 0.07 mmol, 1 eq) in dioxane (5mL) was added (NH₂)₂SO₂ (25 mg, 0.26 mmol, 3.7 eq) and heated to 100° C.for 14 h. The reaction mixture was cooled to room temperature, quenchedby addition of aqueous HCl (1N, 2-3 mL) and concentrated in vacuo.Organic components were extracted with 5% methanol/CH₂Cl₂ (50 mL) andthe organic layer was dried over anhydrous sodium sulfate andconcentrated in vacuo. The crude material was purified by flashchromatography (Combiflash) using 100-200 mesh silica gel eluting with9% methanol/CH₂Cl₂ to obtain compound Xa (18 mg, 48%) as off whitesolid.

¹H NMR (400 MHz, DMSO-d₆) δ 9.12-9.10 (m, 1H), 8.45-8.43 (m, 1H),8.01-7.93 (m, 2H), 7.74-7.70 (m, 2H), 7.61-7.42 (m, 2H), 6.71 (d, J=6Hz, 2H), 5.19-4.99 (m, 1H), 4.73-4.66 (m, 1H), 3.64-3.40 (m, 4H),2.95-2.76 (m, 4H), 1.91-1.68 (m, 3H), 1.68-1.51 (m, 3H), 1.35-1.33 (m,3H), 1.22-0.83 (m, 4H);

LCMS: m/z=536.4 [M+H], RT=2.75 minutes; (Program R1, Column W).

Example 109: Scheme 24 Below is a Representative Synthesis of4-{1-sec-Butyl-7-[((R)-cyclopropyl-quinolin-3-yl-methyl)-amino]-1H-pyrazolo[4,3-d]pyrimidin-5-yl}-piperazine-1-sulfonicAcid Amide) (Xa′)

To the solution of compound IX (30 mg, 0.07 mmol, 1 eq) in DMF (3 mL)were added bromoacetamide (18 mg, 0.13 mmol, 2 eq), K₂CO₃ (27 mg, 0.20mmol, 3 eq) and the resulting mixture was heated to 60° C. for 3 h. Thereaction mixture was cooled to room temperature, diluted with water andthe organic components were extracted with CH₂Cl₂ (50 mL). DCM layer wasthen dried over anhydrous sodium sulfate and concentrated in vacuo. Thecrude material was purified by flash chromatography (Combiflash) using100-200 mesh silica gel eluting with 7% methanol/CH₂Cl₂ to obtaincompound Xa^(/) (25 mg, 75%) as off white solid.

¹H NMR (400 MHz, DMSO-d₆) δ 9.06-9.04 (m, 1H), 8.37-8.36 (m, 1H),7.97-7.95 (m, 1H), 7.91-7.88 (m, 1H), 7.68-7.64 (m, 2H), 7.57-7.51 (m,2H), 7.15-7.07 (2, m H), 5.15-4.91 (m, 1H), 4.75-4.51 (m, 1H), 3.56-3.31(m, 4H), 2.72-2.63 (m, 2H), 2.22-2.17 (m, 4H), 1.78-1.59 (m, 3H),1.54-1.47 (m, 3H), 0.82-0.49 (m, 7H);

LCMS: m/z=514.2 [M+H], RT=2.37 minutes; (Program R1, Column X).

Example 110: Scheme 25 Below is a Representative Synthesisof—4-(1-sec-butyl-7-{[(R)-cyclopropyl(quinolin-3-yl)methyl]amino}-1H-pyrazolo[4,3-d]pyrimidin-5-yl)-N′-cyanopiperazine-1-carboximidamide)(XI)

(4-{1-sec-Butyl-7-[((R)-cyclopropyl-quinolin-3-yl-methyl)-amino]-1H-pyrazolo[4,3-d]pyrimidin-5-yl}-piperazin-1-yl)-phenoxy-methyl-cyanamide(Xb)

To a solution of compound IX (50 mg, 0.11 mmol, 1 eq) in MeCN (5 mL)were added diphenylcyanocarbonimidate (32 mg, 0.13 mmol, 1.2 eq) andtriethylamine (0.02 mL, 0.13 mmol, 1.2 eq) and the resulting mixture wasstirred at 60° C. for 14 h. The reaction mixture was cooled to RT,concentrated in vacuo to obtain compound Xb as light brown sticky solidwhich was used in the next step without further purification.

LCMS: m/z=601.3 [M+H], RT=3.08 minutes; (Program R1, Column W).

4-(1-sec-butyl-7-{[(R)-cyclopropyl(quinolin-3-yl)methyl]amino}-1H-pyrazolo[4,3-d]pyrimidin-5-yl)-N′-cyanopiperazine-1-carboximidamide(XI)

To a solution of compound Xb (65 mg, 0.11 mmol, 1 eq) in methanol (3 mL)was added NH₄OH (0.4 mL) and the resulting mixture was refluxed for 5 h.The reaction mixture was cooled to RT, concentrated in vacuo. The crudematerial was purified by flash chromatography (Combiflash) using 100-200mesh silica gel eluting with 5% methanol/CH₂Cl₂ to obtain compound XI(25 mg, 43% for 2 steps) off white solid.

¹H NMR (400 MHz, DMSO-d₆) δ 9.11-9.10 (m, 1H), 9.09-8.40 (m, 1H),8.00-7.93 (m, 2H), 7.73-7.70 (m, 1H), 7.63-7.58 (m, 3H), 7.13-7.12 (m,2H), 5.11-5.08 (m, 1H), 4.76-4.64 (m, 1H), 3.50-3.33 (m, 8H), 1.91-1.66(m, 3H), 1.56-1.50 (m, 3H), 0.77-0.50 (m, 7H);

LCMS: m/z=524.6 [M+H], RT=2.72 minutes; (Program R1, Column W).

Example 111: Scheme 25 was Also Used to Synthesize4-(1-sec-butyl-7-{[(R)-methyl(quinolin-3-yl)methyl]amino}-1H-pyrazolo[4,3-d]pyrimidin-5-yl)-N′-cyanopiperazine-1-carboximidamide(See Table 5 Below)

TABLE 5 Example No. Molecular Structure LCMS 111

LCMS: m/z = 498.0 [M + H], RT = 2.47 minutes; (Program R1, Column W).

Example 112: Scheme 26 Below is a Representative Synthesisof—4-{1-sec-Butyl-7-[((R)-cyclopropyl-quinolin-3-yl-methyl)-amino]-1H-pyrazolo[4,3-d]pyrimidin-5-yl}-piperazine-1-carboxamidine)(XII)

[[(E)-tert-Butoxycarbonylimino]-(4-{1-sec-butyl-7-[((R)-cyclopropyl-quinolin-3-yl-methyl)-amino]-1H-pyrazolo[4,3-d]pyrimidin-5-yl}-piperazin-1-yl)-methyl]-carbamicacid tert-butyl Ester (Xc)

To the solution of IX (50 mg, 0.11 mmol, 1 eq) in NMP (5 mL) were added(tert-butoxycarbonylimino-pyrazol-1-yl-methyl)-carbamic acid tert-butylester (34 mg, 0.11 mmol, 1 eq) and DIPEA (0.04 mL, 0.22 mmol, 2 eq) andthe resulting mixture was stirred at 23° C. for 16 h. The reactionmixture was diluted with water (10 mL) and the organic components wereextracted with CH₂Cl₂ (50 mL). The organic layer was dried over anhyd.sodium sulfate and concentrated in vacuo. The crude residue was purifiedby flash chromatography (Combiflash) using 100-200 mesh silica geleluting with 2% MeOH/CH₂Cl₂ to obtain compound Xc (100 mg) as brownsolid.

LCMS: m/z=699.6 [M+H], RT=3.22 minutes; (Program R1, Column W).

4-{1-sec-Butyl-7-[((R)-cyclopropyl-quinolin-3-yl-methyl)-amino]-1H-pyrazolo[4,3-d]pyrimidin-5-yl}-piperazine-1-carboxamidine(XII)

To a solution of compound Xc (100 mg, 0.14 mmol, 1 eq) in CH₂Cl₂ (10 mL)was added TFA (0.11 mL, 1.43 mmol, 10 eq) under ice-cold condition andthe resulting mixture was stirred at 23° C. for 5 h. The reactionmixture was concentrated in vacuo. The residue was diluted with toluene(3 mL) and further concentrated in vacuo. The residue was diluted withaq. NaHCO₃ solution [5 mL] and the organic components were extractedwith 5% MeOH/CH₂Cl₂ (50 ml). The organic layer was concentrated in vacuoto obtain compound XII (40 mg, 57%) as yellow solid (XII).

¹H NMR (400 MHz, DMSO-d₆) δ 9.10-9.08 (m, 1H), 8.42-8.40 (m, 1H),8.01-7.93 (m, 2H), 7.74-7.71 (m, 2H), 7.65-7.60 (m, 2H), 7.40-7.20 (m,3H), 5.09-5.08 (m, 1H), 4.80-4.73 (m, 1H), 3.60-3.32 (m, 8H), 1.90-1.60(m, 3H), 1.57-1.50 (m, 3H), 0.77-0.57 (m, 7H);

LCMS: m/z=499.3 [M+H], RT=2.10 minutes; (Program R1, Column W).

Example 113: Scheme 27 Below is a Representative Synthesis of2-{4-[1-sec-Butyl-7-((R)-1-quinolin-3-yl-ethylamino)-1H-pyrazolo[4,3-d]pyrimidin-5-yl]-pyrazol-1-yl}-ethanol

Intermediate VIIa was synthesized in a similar manner to that of VII.

(1-sec-Butyl-5-{1-[2-(tert-butyl-dimethyl-silanyloxy)-ethyl]-1H-pyrazol-4-yl}-1H-pyrazolo[4,3-d]pyrimidin-7-yl)-((R)-1-quinolin-3-yl-ethyl)-amine(VIIIa)

To a stirred and degassed [with Ar] solution of VIIa, as prepared inScheme 22 (200 mg, 0.53 mmol, 1 eq) in a mixture of toluene (14 ml) andEtOH (6 ml) was added Cs₂CO₃ (513 mg, 1.58 mmol, 3 eq), compound VIIb(278 mg, 0.79 mmol, 1.5 eq) and the mixture was again degassed with Arfor 15 min. To the reaction mixture was added Pd(PPh₃)₄ (32 mg, 0.05mmol, 0.1 eq) and the resulting mixture was stirred at 90° C., under Aratmosphere for 16 h. The reaction mixture was cooled to RT, filteredthrough Celite and the filtrate was concentrated under reduced pressureto obtain crude product VIIIa (300 mg) as off white solid. This was usedfor the next step without further purification.

2-{4-[1-sec-Butyl-7-((R)-1-quinolin-3-yl-ethylamino)-1H-pyrazolo[4,3-d]pyrimidin-5-yl]-pyrazol-1-yl}-ethanol

To a stirred solution of VIIIa (300 mg, 0.53 mmol, 1 eq) in THF (15 ml)was added TBAF (1 ml, 1M in THF) and the reaction mixture was stirred at23° C. for 2 h. The reaction mixture was concentrated under reducedpressure to obtain crude product which was purified by flashchromatography (Combiflash) using 100-200 mesh silica gel eluting with10% MeOH/CH₂Cl₂ to obtain compound CE01111 (20 mg) as off white solid.

LCMS: m/z=457.4 [M+H], RT=3.21 minutes; (Program R1, Column W).

Examples 114-123: The 10 compounds shown in Table 6 below weresynthesized using the Scheme 27.

Example No. Molecular Structure LCMS 114

LCMS: m/z = 453.4 [M + H], RT = 2.98 minutes; (Program R1, Column W).115

LCMS: m/z = 467.4 [M + H], RT = 3.41 minutes; (Program R1, Column W).116

LCMS: m/z = 485.2 [M + H], RT = 3.47 minutes; (Program R1, Column W).117

LCMS: m/z = 481.2 [M + H], RT = 2.86 minutes; (Program P1, Column V).118

LCMS: m/z = 499.2 [M + H], RT = 3.00 minutes; (Program R1, Column W).119

LCMS: m/z = 439.2 [M + H], RT = 3.37 minutes; (Program R1, Column W).120

LCMS: m/z = 496.0 [M + H], RT = 3.44 minutes; (Program R1, Column W).121

LCMS: m/z = 483.2 [M + H], RT = 3.28 minutes; (Program R1, Column W).122

LCMS: m/z = 475.2 [M + H], RT = 3.33 minutes; (Program R1, Column W).123

LCMS: m/z = 462.2 [M + H], RT = 2.84 minutes; (Program R1, Column W).

The compound of Example 118 was synthesized via the Suzuki reaction ofVIIa as shown in Scheme 27 with4-(4,4,5,5-Tetramethyl-[1,3,2]dioxaborolan-2-yl)-3,6-dihydro-2H-pyridine-1-carboxylicacid tert-butyl ester, followed by further reactions as steps 8 and 9 asshown in Scheme 21, with the final double bond reduction carried out byhydrogenation with 10% Pd/C in EtOH.

Example 124: Scheme 28 Below is a Representative Synthesis of1-sec-Butyl-7-[((R)-cyclopropyl-quinolin-3-yl-methyl)-amino]-1H-pyrazolo[4,3-d]pyrimidine-5-carboxylicAcid Cyclopropylamide

1-sec-Butyl-7-[((R)-cyclopropyl-quinolin-3-yl-methyl)-amino]-1H-pyrazolo[4,3-d]pyrimidine-5-carboxylicAcid Methyl Ester (VIII)

To a stirred solution of VII as prepared in Scheme 21 (500 mg, 1.23mmol, 1 eq) in MeOH (2 ml) were added PdCl₂(dppf).DCM (100 mg, 0.123mmol, 0.1 eq) and DIPEA (1.1 ml, 6.16 mmol, 5 eq) and the mixture wasdegassed with Ar for 15 min. the resulting mixture was subjected toreaction in Parr autoclave under 50 psi CO pressure at 90° C. for 16 h.The reaction mixture was cooled to RT, filtered through Celite and thefiltrate was concentrated under reduced pressure to obtain crude productwhich was purified by flash chromatography (Combiflash) using 100-200mesh silica gel eluting with 65% EtOAc/hexane to obtain compound VIII(440 mg, 84%).

¹H NMR (400 MHz, DMSO-d₆) δ 9.18-9.13 (m, 1H), 8.53-8.50 (m, 1H), 8.25(m, 1H), 8.06-7.94 (m, 3H), 7.74-7.70 (m, 1H), 7.62-7.55 (m, 1H), 5.20(m, 1H), 4.95-4.80 (m, 1H), 3.82-3.81 (m, 3H), 1.98-1.80 (m, 3H),1.60-1.55 (m, 3H), 0.75-0.50 (m, 7H);

LCMS: m/z=431.2 [M+H], RT=3.78 minutes; (Program R1, Column W).

1-sec-Butyl-7-[((R)-cyclopropyl-quinolin-3-yl-methyl)-amino]-1H-pyrazolo[4,3-d]pyrimidine-5-carboxylicAcid (IX)

To a stirred solution of VIII (440 mg, 1.023 mmol, 1 eq) in THF (8 ml)and water (4 ml) was added LiOH.H₂O (86 mg, 2.05 mmol, 2 eq) and themixture was stirred at 23° C. for 4 h. The reaction mixture wasconcentrated under reduced pressure and diluted with water. The aqueouslayer was acidified with dilute HCl and the organic components wereextracted into MeOH/CH₂Cl₂ to obtain compound IX (370 mg, crude), whichwas used for the next step without further purification.

¹H NMR (400 MHz, DMSO-d₆) δ 9.30-9.25 (m, 1H), 8.78-8.72 (m, 1H), 8.25(s, 1H), 8.12-8.00 (m, 3H), 7.81 (m, 1H), 8.70 (m, 1H), 5.21 (m, 1H),5.01-4.92 (m, 1H), 2.00-1.70 (m, 3H), 1.60-1.54 (m, 3H), 0.80-0.50 (m,7H);

LCMS: m/z=417.2 [M+H], RT=3.29 minutes; (Program R1, Column W).

1-sec-Butyl-7-[((R)-cyclopropyl-quinolin-3-yl-methyl)-amino]-1H-pyrazolo[4,3-d]pyrimidine-5-carboxylicAcid Cyclopropylamide

To a stirred solution of IX (50 mg, 0.12 mmol, 1 eq) in DMF (3 ml) wereadded cyclopropyl amine (8 mg, 0.144 mmol, 1.2 eq) DIPEA (23 mg, 0.18mmol, 1.5 eq) and1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxid hexafluorophosphate (HATU) (59 mg, 0.16 mmol, 1.3 eq) and stirredat 23° C. for 5 h. The reaction mixture was diluted with water and theorganic components were extracted with CH₂Cl₂ to obtain crude product,which was purified by flash chromatography (Combiflash) using 100-200mesh silica gel eluting with 45% MeOH/CH₂Cl₂ to obtain compound1-sec-Butyl-7-[((R)-cyclopropyl-quinolin-3-yl-methyl)-amino]-1H-pyrazolo[4,3-d]pyrimidine-5-carboxylicacid cyclopropylamide (440 mg, 84%).

¹H NMR (400 MHz, DMSO-d₆) δ 9.17-9.16 (m, 1H), 8.50-8.49 (m, 1H),8.21-8.20 (m, 1H), 8.19-8.12 (m, 1H), 8.01-7.90 (m, 3H), 7.74-7.68 (m,1H), 7.62-7.57 (m, 1H), 5.30-5.20 (m, 1H), 5.05-4.90 (m, 1H), 2.80-2.70(m, 1H), 2.00-1.90 (m, 3H), 1.63-1.55 (m, 3H), 1.25 (m, 1H), 0.80-0.45(m, 10H);

LCMS: m/z=456.4 [M+H], RT=4.13 minutes; (Program R1, Column W).

Example 125: Scheme 29 is a Representative Synthesis of[1-sec-Butyl-5-(5-methyl-oxadiazol-2-yl)-1H-pyrazolo[4,3-d]pyrimidin-7-yl]-((R)-cyclopropyl-quinolin-3-yl-methyl)-amine

1-sec-Butyl-7-[((R)-cyclopropyl-quinolin-3-yl-methyl)-amino]-1H-pyrazolo[4,3-d]pyrimidine-5-carboxylicAcid N′-acetyl-hydrazide (X)

To a stirred solution of IX as prepared in Scheme 28 (160 mg, 0.38 mmol,1 eq) in DMF (8 ml) was added acetic acid hydrazide (34 mg, 0.46 mmol,1.2 eq) DIPEA (73 mg, 0.58 mmol, 1.5 eq) and HATU (190 mg, 0.50 mmol,1.3 eq) and stirred at 23° C. for 16 h. The reaction mixture was dilutedwith water and the organic components were extracted with CH₂Cl₂ toobtain crude product which was purified by flash chromatography(Combiflash) using 100-200 mesh silica gel eluting with 8% MeOH/CH₂Cl₂to obtain compound X (180 mg, 99%).

LCMS: m/z=473.0 [M+H], RT=3.34 minutes; (Program R1, Column W).

[1-sec-Butyl-5-(5-methyl-[1,3,4]oxadiazol-2-yl)-1H-pyrazolo[4,3-d]pyrimidin-7-yl]-((R)-cyclopropyl-quinolin-3-yl-methyl)-amine

Compound IX (180 mg, 0.38 mmol, 1 eq) was taken in POCl₃ (3 ml) andrefluxed for 3 h. The reaction mixture was then cooled to RT, quenchedwith aq. NaHCO₃ and the organic components were extracted with CH₂Cl₂ toobtain crude product which was purified by flash chromatography(Combiflash) using 100-200 mesh silica gel eluting with 2% MeOH/CH₂Cl₂followed by prep TLC (60% EtOAc/hexane) to obtain compound CE01108 (15mg, 9%).

¹H NMR (400 MHz, DMSO-d₆) δ 9.22-9.18 (m, 1H), 8.61-8.58 (m, 1H),8.28-8.27 (m, 1H), 8.18-8.15 (m, 1H), 8.00-7.93 (m, 2H), 7.74-7.69 (m,1H), 7.61-7.57 (m, 1H), 5.28 (m, 1H), 4.90-4.80 (m, 1H), 2.59 (m, 2H),2.00-1.80 (m, 3H), 1.62-1.56 (m, 3H), 0.76-0.56 (m, 6H);

LCMS: m/z=455.3 [M+H], RT=3.07 minutes; (Program P1, Column V).

Example 126: Scheme 30 is a Representative Synthesis of1-{1-sec-Butyl-7-[((R)-cyclopropyl-quinolin-3-yl-methyl)-amino]-1H-pyrazolo[4,3-d]pyrimidin-5-yl}-1H-[1,2,3]triazole-4-carboxylicAcid Amide

(5-Azido-1-sec-butyl-1H-pyrazolo[4,3-d]pyrimidin-7-yl)-((R)-cyclopropyl-quinolin-3-yl-methyl)-amine(VIII)

To a stirred solution of VII as prepared in Scheme 21 (100 mg, 0.25mmol, 1 eq) in DMF (2 ml) in a sealed tube were added K₂CO₃ (68 mg, 0.49mmol, 2 eq) and NaN₃ (32 mg, 0.49 mmol, 2 eq) and stirred at 160° C. for2 days. The reaction mixture was cooled to RT, diluted with water andthe organic components were extracted with EtOAc to obtain crude productwhich was purified by flash chromatography (Combiflash) using 100-200mesh silica gel eluting with 70% EtOAc/hexane to obtain compound VIII(80 mg, 79%).

LCMS: m/z=414.2 [M+H], RT=3.80 minutes; (Program P1, Column V).

1-{1-sec-Butyl-7-[((R)-cyclopropyl-quinolin-3-yl-methyl)-amino]-1H-pyrazolo[4,3-d]pyrimidin-5-yl}-1H-[1,2,3]triazole-4-carboxylicAcid Ethyl Ester (IX)

To a stirred solution of VIII (80 mg, 0.19 mmol, 1 eq) in MeOH (5 ml)were added a solution of sodium ascorbate (4 mg, 0.019 mmol, 0.1 eq,dissolved in 0.3 ml water), Cu(OAc)₂.H₂O (4 mg, 0.019 mmol, 0.1 eq) andethyl propiolate (0.06 ml, 0.58 mmol, 3 eq) and the resulting mixturewas stirred at 23° C. for 16 h. The reaction mixture was concentrated invacuo and purified by flash chromatography (Combiflash) using 100-200mesh silica gel eluting with 84% EtOAc/hexane to obtain compound IX (30mg, 30%).

LCMS: m/z=512.2 [M+H], RT=4.32 minutes; (Program P1, Column V).

1-{1-sec-Butyl-7-[((R)-cyclopropyl-quinolin-3-yl-methyl)-amino]-1H-pyrazolo[4,3-d]pyrimidin-5-yl}-1H-[1,2,3]triazole-4-carboxylicAcid Amide

To a stirred solution of IX (30 mg, 0.058 mmol, 1 eq) in MeOH (3 ml) ina sealed tube was added aqueous ammonium hydroxide (2 ml) and themixture was stirred at 23° C. for 16 h. The reaction mixture wasconcentrated in vacuo and purified by prep TLCl eluting with 70%EtOAc/hexane to obtain compound CE01091 (7 mg, 24%).

¹H NMR (400 MHz, DMSO-d₆) δ 9.22-9.18 (m, 1H), 9.13-9.09 (m, 1H),8.60-8.56 (m, 1H), 8.38-8.34 (m, 1H), 8.25-8.24 (m, 1H), 8.03-7.92 (m,3H), 7.73-7.69 (m, 1H), 7.61-7.58 (m, 2H), 5.26 (m, 1H), 5.14-5.03 (m,1H), 2.00-1.70 (m, 43H), 1.63-1.57 (m, 3H), 0.79-0.57 (m, 6H);

LCMS: m/z=483.2 [M+H], RT=2.93 minutes; (Program P1, Column V).

Example 127: Scheme 31 is a Representative Synthesis of(1-sec-Butyl-5-pyridazin-3-yl-1H-pyrazolo[4,3-d]pyrimidin-7-yl)-((R)-cyclopropyl-quinolin-3-yl-methyl)-amine

(1-sec-Butyl-5-pyridazin-3-yl-1H-pyrazolo[4,3-d]pyrimidin-7-yl)-((R)-cyclopropyl-quinolin-3-yl-methyl)-amine

To a stirred solution of VII prepared in Scheme 21 [for CE00976] (100mg, 0.25 mmol, 1 eq) in DMF (5 ml) in a sealed tube was added3-(tributyltin)pyridazine (182 mg, 0.49 mmol, 2 eq) and the mixture wasdegassed with Ar for 30 min. To this reaction mixture was addedPd(PPh₃)₄ (30 mg, 0.025 mmol, 0.1 eq) and degassing was repeated with Arfor another 10 min. The reaction mixture was then heated at 120° C. for18 h. The reaction mixture was cooled to RT, filtered through Celite andthe filtrate was concentrated under reduced pressure to obtain crudeproduct which was purified by flash chromatography (Combiflash) using100-200 mesh silica gel eluting with 70% EtOAc/hexane to obtain compoundCE01099 (40 mg, 36%).

¹H NMR (400 MHz, DMSO-d₆) δ 9.90-9.86 (m, 1H), 9.32-9.28 (m, 1H),9.26-9.22 (m, 1H), 8.58 (m, 1H), 8.32-8.28 (m, 2H), 8.15-8.09 (m, 1H),8.00-7.94 (m, 2H), 7.75-7.65 (m, 1H), 7.60-7.55 (m, 1H), 5.28 (m, 1H),5.16-5.05 (m, 1H), 2.00-1.70 (m, 3H), 1.67-1.58 (m, 3H), 0.79-0.69 (m,6H);

LCMS: m/z=451.6 [M+H], RT=3.98 minutes; (Program R1, Column W).

Example 128: In Vitro Studies

A. Cloning

The FLIPR® assay utilizes cells which express human or rat P2X3 orP2X2/3 receptors. Recombinant cells expressing hP2X3 (Cat #6188) andhP2X2/3 (Cat #6179) were procured from Chantest Corp. Rat P2X2 (NCBIAccession No: U14414) was amplified by PCR from PC12 cDNA (a rat adrenalmedulla cell line). The PCR product obtained containing the proteincoding sequence of rat P2X2 was cloned into EcoRV-digested anddephosphorylated vector plRES-puro3 within the multiple cloning site(MCS). See, FIG. 1A.

Rat P2X3 (NCBI Accession No: X91167) was amplified by PCR from rat braincDNA. The PCR product obtained containing the protein coding sequence ofrat P2X3 was cloned into EcoRV-digested and dephosphorylated vectorpCDNA-Hygro within the multiple cloning site (MCS) (FIG. 1B). Rat P2X3cloned into pcDNA-Hygro was then subcloned into pcDNA-5/TO at HindIII(5′) and XhoI (3′) sites within the multiple cloning site (MCS) of thevector (FIG. 1C).

All the constructs yielding the recombinant vector DNA were used fortransfection and generation of the cell lines, after sequenceverification.

B. Development of Recombinant TRex293 Cells Expressing rP2X2/3 andCHO-TRex Cells Expressing rP2X3

Transfection was carried out using super-coiled constructs (purifiedusing QIAGEN kit) in antibiotic free, serum free DMEM usingLipofectamine® 2000 (Invitrogen) transfection agent. The DNA constructsrat P2X2 in pIRES-Puro3 and rat P2X3 in pCDNA5/TO were co-transfectedinto TRex293 cells in order to generate the rP2X2/3 stable line. 50μg/mL hygromycin (Invitrogen) and 0.5 μg/mL puromycin (Fermentek) wereused for selection of stable clones of rP2X2/3. Rat P2X3 in pCDNA5/TODNA construct was transfected to CHO-TRex cells to generate the rP2×3stable line and 500 μg/mL hygromycin (Invitrogen) was used as selectionantibiotic. Transfected stable colonies were then functionally verifiedand robust clones suitable for assay were clonally purified throughdilutions.

C. Assay Protocols

(i) Intracellular Calcium Assay Protocol for Screening Compounds

Cryo-vial containing 6×10⁶ cells (human P2X3-HEK/humanP2X2/3-TRex293/rat P2X2/3-TRex293/rat P2X3 TRex-CHO) was thawed in a 37°C. water bath. Cells were suspended in 20 mL of respective cell platingmedia (See annexure for composition) in a 50 mL centrifuge tube. Thecell viability was checked with the help of Trypan Blue dye. Uponwashing, cells were plated in a black 384-well clear bottomed, sterilepoly-D-lysine coated plate such that, each well contained 10,000 cells(15,000/well for hP2X3) in 30 μL cell plating media. The plate wasincubated in a 5% CO₂ incubator at 37° C. for 24 h.

The next day, prior to the assay, the cell plating media was removedfrom each well by decanting and gentle tapping. Thirty μL of FLIPRCalcium 4 dye solution was added to each well. The plate was incubatedat 37° C. for 45 min (60 min for hP2X3). The plate was next equilibratedat room temperature for 15 minutes before placing it in a 384 well FLIPRfor the assay.

Compounds were dissolved in DMSO and serially diluted following 11 pointhalf log (3.16 fold) dilution with a starting concentration of 2 mM.Dilutions were mixed with assay buffer just before performing the assay.

Compounds were added to the respective wells of the assay-ready cellplate with the help of the FLIPR and fluorescence readings were capturedfor 5 min to observe any possible agonistic property of the compounds.The plate was then incubated at room temperature for 15 min. The cellswere stimulated with respective agonist EC₇₅ concentration and thefluorescence readings were captured for another 5 min by FLIPR. Thedifference in fluorescence readings in presence of the compounds werecompared with that of the control wells (wells having no compound) tocalculate the inhibitory potency of the compounds. The IC₅₀ values ofthe compounds were determined using the Graph pad Prism software.

(ii) Cell Plating Media for hP₂X₃-HEK and hP₂X_(2/3)-TRex293 Cells

-   -   DMEM/F12 (1:1) HAM media (Invitrogen; Cat #11039)    -   1×NEAA (Invitrogen; Cat #11140)    -   25 mM HEPES (Invitrogen; Cat #15630)    -   1 mM sodium pyruvate (Invitrogen; Cat #11360)    -   10% tetracycline negative FBS (PAA; Cat #A15-209)    -   1 μg/mL doxycycline (Clontech; Cat #63131) [for hP2X2/3-TRex293        cells only]

(iii) Cell Plating Media for rP₂X_(2/3)-TRex293 Cells

-   -   DMEM media (Invitrogen; Cat #11965)    -   25 mM HEPES (Invitrogen; Cat #15630)    -   10% tetracycline negative FBS (PAA; Cat #A15-209)    -   1 μg/mL Doxycycline (Clontech; Cat #63131)

(iv) Cell Plating Media for rP₂X₃-CHOTRex Cells

-   -   F-12 nutrient mixture (HAM) 1× (Invitrogen; Cat #11765)    -   1× Glutamax™ (Invitrogen; Cat #35050)    -   10% tetracycline negative FBS (PAA; Cat #A15-209)    -   1 μg/mL doxycycline (Clontech; Cat #63131)

(v) Assay Buffer Composition

-   -   HBSS (Invitrogen Cat #14025)    -   20 mM HEPES (Invitrogen Cat #15630)    -   0.01% F127 (Sigma Cat #P2443)    -   1.8 mM CaCl₂ (Sigma Cat #C₅₀₈₀)    -   pH adjusted to 7.4

(vi) Dye Solution Composition

-   -   1× FLIPR Calcium 4 dye in assay buffer (Molecular devices Cat        #R8141)    -   1.8 mM Probenecid (Sigma Cat #P8761)    -   pH adjusted to 7.4

Data from the P2X₃ and P2X_(2/3) FLIPR assays for compounds of formula(I) are shown in Table 7 below.

TABLE 7 Example hP2X₃ hP2X_(2/3) 1 A D 2 B 3 B C 4 A D 5 A D 6 B C 7 B D8 A 9 B 10 A A 11 A C 12 A C 13 A C 14 B D 15 A C 16 A C 17 B 18 B C 19A B 20 B 21 A C 22 A A 23 A C 24 A C 25 A C 26 B 27 A 28 B 29 A C 30 A C31 A C 32 A C 33 A C 34 A A 35 A B 36 B 37 A 38 B 39 B C 40 A B 41 B 42A B 43 A C 44 A A 45 A C 46 A B 47 A C 48 A C 49 A C 50 B 51 A A 52 A C53 B 54 B 55 A B 56 A B 57 A B 58 A C 59 A B 60 A B 61 A B 62 A B 63 B64 A C 65 A B 66 A B 67 B 68 A B 69 A A 70 A A 71 B 72 A C 73 B 74 A B75 A C 76 B C 77 A C 77 B C 78 B 79 B C 80 A C 81 A B 82 A C 83 A C 84 AC 85 A B 86 A A 88 A C 89 A B 90 A C 91 A C 93 A C 94 A C 95 B 96 A D 97A 98 A C 99 A C 100 A C 101 B C 102 A D 103 A D 104 A D 105 A C 106 A C107 A 108 A C 109 B 110 A A 111 A A 112 A A 113 A C 114 A A 115 A B 116A A 117 A C 118 A C 119 A C 120 B 121 A B 122 A B 123 A C 123 A B 124 AC 125 A C 126 A C 127 A C A: IC₅₀ = 1-100 nM B: IC₅₀ = >100-1000 nM C:IC₅₀ = >1000-10,000 nM D: IC₅₀ >10,000 nM

Example 129: In Vivo Thermal Hyperalgesia (Hargreaves Test) Studies inthe Rat

Male Sprague Dawley rats of young adult age group and body weight rangeof 180-200 g were included in the study. Animals were housed under a 12h light/dark cycle with food and water ad libitum. The animals underwentacclimatization with the observation chambers of the Hargreaves'apparatus for two days, twice daily for 45-60 min each time prior toinitiation of study. Animals were also habituated to the apparatus for15-30 min before each testing. Thermal hyperalgesia was assessed usingthe rat plantar test (Ugo Basile, Italy) following a modified method ofHargreaves (1988), “A new and sensitive method for measuring thermalnociception in cutaneous hyperalgesia”, Pain 32: 77-88, the entiredisclosure of which is herein incorporated by reference.

For measurement of paw withdrawal latency (PWL) values the rats wereexposed to a mobile infrared heat source applied directly below theplantar surface of the rat hind paw. The PWL was defined as the time inseconds taken by the rat to remove its hind paw from the heat source.Thirty three percent IR of the instrument was used to measure PWL.Animals showing basal response between 8-14 sec on an untreated paw wereincluded in the study. A cut off point of 20 sec was used to preventtissue damage.

Following basal readout of PWL values to the thermal stimulus (PWLmeasurements described previously), 50 μL of complete Freund's Adjuvant(CFA—1 mg/mL suspension—Sigma, USA, Cat #F5881) was injectedsubcutaneously into the plantar surface of the right hind (ipsilateral)paw of animals under light isoflurane anesthesia. A one mL syringe and26 g^(1/2)-inch needle was used for the injection. CFA suspension wasmixed thoroughly before each injection. Light pressure was applied tothe injection site for 10 s immediately after the needle was removedfrom the paw to prevent any leaking out of adjuvant oil from theinjection site. The rats were then returned to their housing to recoverand kept in soft bedding.

Next day (day 1) after 20-22 h of CFA injection, PWL of animals wererecorded. Mean of three readings are taken as PWL recording ofipsilateral paw of each animal for pre and post CFA basal readout.Animals with PWL values of <6 sec on day 1 post CFA injection wereconsidered hyperalgesic and selected for randomization into treatmentgroups and further test sessions following a single blind protocol.

In the test session, PWL was assessed at 1 h post oral dosing of CE testarticle, vehicle (20% polyethylene glycol, 1% Tween™ 80, 79% water) andnaproxen (positive control).

Statistical analysis was done with One-way ANOVA followed by Dunnett'smultiple comparison post-test. Post treatment PWL values were comparedwith pre-treatment PWL values and p<0.05 was considered statisticallysignificant. Each group was typically comprised of 8 animals.

Compounds of the present disclosure were found to be efficacious in thisthermal hyperalgesia pain model. The compound of Example 19 was found toprovide 36% reversal (p<0.01) to pre-treatment PWL value at 1 hourfollowing a dose of 60 mg/kg po. The compound of Example 10 was found toprovide 32% reversal (p<0.01) to pre-treatment PWL value at 1 hourfollowing a dose of 30 mg/kg po.

Example 130: Formalin Induced Pain (Automated Nociception Analyzer Test)in the Rat

Male Sprague Dawley rats of young adult age group and body weight rangeof 200-250 g are included in the study. Animals are housed under a 12 hlight/dark cycle with food and water ad libitum. Animals areacclimatized in the observation chambers of Automated NociceptionAnalyzer (ANA) for 45-60 min, twice daily for two days prior to thestudy day. On the day of the study, metal bands are glued to the plantarsurface of the right hind paw of each animal enrolled in the study setand kept in plastic observation chambers for 10-15 min. Formalininjection is done in the animals after 0.5 or 1 h of oral treatment witha compound of formula (I) in 20% polyethylene glycol, 1% Tween™ 80reagent, 79% water (the “test article”) or vehicle (20% polyethyleneglycol, 1% Tween™ 80 reagent, 79% water). Formalin injection of theanimals is done with 50 μL of 2.5% formalin (freshly prepared fromformaldehyde solution, Sigma, USA, Cat #F8775) injected subcutaneouslyin to the dorsum of right hind paw. Animals are placed back to theirrespective recording chambers of ANA immediately after injection. Flinchcount data for each animal is recorded from 1 to 60 min post formalininjection, using ANA motion analysis software. The study is analyzed in2 phases, the early phase extended from 0-10 min and second phaseextended from 11-60 min post formalin injection. The data is collectedin 5 min time bins and the counts of each bin are added up for totalcount of the phase.

Statistical analysis is done with unpaired t test. Comparison is donebetween total count of treatment groups and vehicle group and p<0.05 isconsidered statistically significant. Eight animals are typically usedin each of test article and vehicle treated groups.

Example 131: Acetic Acid Induced Writhing Test in Mice

Swiss albino mice of 30-40 g are included in the study. The mice aregiven an intraperitoneal injection of 0.7% v/v acetic acid solution atan injection volume of 10 mL/kg, 30 min after oral administration of acompound of formula (I) (the “test article”) or vehicle. The testarticles are administered at doses between 20 and 60 mg/kg. The mice areplaced individually into glass chambers. The number of writhes producedin these animals is counted for 15 min following acetic acidadministration. For scoring purposes writhing is indicated by stretchingof the abdomen with simultaneous stretching of at least one hind limb.

Statistical analysis is done with One-way ANOVA followed by Dunnett'smultiple comparison tests. Comparison is done between treatment groupsand vehicle with respect to total number of writhes and p<0.05 isconsidered statistically significant. Each group is typically comprisedof 6 animals.

Example X: Citric Acid Induced Cough in the Guinea Pig

Compounds of formula (I) are tested for their ability to suppress coughby using the guinea pig model of citric acid induced cough [Kamei,Takahashi, Yoshikawa, and Saitoh, Eur J Pharmacol 528, p 158-161 (2005);Kamei and Takahashi, Eur J Pharmacol 547, p 160-164, (2006); Lewis etal., Pulm Pharmacol Ther. 20, p 315-3³³ (2007); Leung et al., Cough 3, p10-14 (2007), the entire disclosures of which are herein incorporated byreference]. In this model, guinea pigs are administered a compound offormula (I) (the “test article”) or vehicle, orally or byintraperitoneal injection, 30 to 60 minutes before the cough assessment.The test article is administered at doses ranging from 1 mg/kg to 100mg/kg. For the cough assessment, animals are exposed to 0.4 M citricacid aerosol inhalation for 10 minutes, and the cough response isrecorded as the number of coughs per 10 minutes, as determined by anexperienced investigational observer and/or by microphone. In avariation of this model, the animals are sensitized by treatment withinhaled histamine or alpha, beta-methylene ATP prior to the inhaledcitric acid treatment.

All publications cited in this specification are incorporated herein byreference. While the present disclosure has been described withreference to particular embodiments, it will be appreciated thatmodifications can be made without departing from the spirit of thepresent disclosure. Such modifications are intended to fall within thescope of the appended claims.

What is claimed is:
 1. A compound of the structure of formula (I):

and prodrugs, enantiomers or pharmaceutically acceptable salts thereof,wherein: R¹ is H, halogen, C₁-C₆ alkyl, C₃-C₆ cycloalkyl, CN or CONH₂;R² is none, optionally substituted C₁-C₆ alkyl or aryl; R³ is none, H,optionally substituted C₁-C₆ alkyl, C₃-C₆ cycloalkyl or heterocycle,wherein when R² is none, R³ is optionally substituted C₁-C₆ alkyl, C₃-C₆cycloalkyl or heterocycle, and wherein when R³ is none, R² is optionallysubstituted C₁-C₆ alkyl or aryl; R⁴ is optionally substituted quinoline;R⁵, R⁶ are independently H, C₁-C₆ alkyl or C₃-C₆ cycloalkyl; and R⁷ isCONHR⁸, optionally substituted morpholine, optionally substitutedpiperazine or optionally substituted heteroaryl, wherein R⁸ is H, alkylor cycloalkyl.
 2. A compound according to claim 1, wherein R¹ is H.
 3. Acompound according to claim 1, wherein R² is optionally substitutedC₁-C₆ alkyl.
 4. A compound according to claim 1, wherein R³ is H.
 5. Acompound according to claim 1, wherein R¹ is halogen, C₁-C₆ alkyl, orCN.
 6. A compound according to claim 1, wherein R⁷ is selected from thegroup consisting of:

wherein: X is O or H, H; R⁹ is H, optionally substituted C₁-C₆ alkyl,CO(C₁-C₆ alkyl), CONH₂, C(NH)NH₂, C(N—CN)NH₂ or SO₂NH₂; R¹⁰ is H oralkyl optionally substituted with —OH; R¹¹ is H, CH₂—CONH₂ or alkyloptionally substituted with —OH or halogen; and R¹² is H or CONH₂.
 7. Acompound according to claim 1, wherein R¹ and R³ are H.
 8. A compoundaccording to claim 1, wherein R⁵ and R⁶ are independently H or C₃-C₆cycloalkyl.
 9. A compound according to claim 1, wherein R¹ and R³ are Hand R² is optionally substituted C₁-C₆ alkyl.
 10. A compound accordingto claim 1, wherein R¹ and R³ are H, R² is optionally substituted C₁-C₆alkyl, R⁵ and R⁶ are independently H or C₁-C₆ alkyl or are independentlyH and C₃-C₆ cycloalkyl, and R⁷ is

wherein X is H, H and R⁹ is CONH₂.
 11. A pharmaceutical compositioncomprising a compound of claim 1 and a pharmaceutically acceptableexcipient.
 12. A kit comprising a compound of claim
 1. 13. A kitcomprising the pharmaceutical composition of claim
 11. 14. A method forregulating one or both of P2X₃ or P2X_(2/3) receptors in a subject, saidmethod comprising administering a therapeutically effective amount of acompound of claim 1 to the subject.
 15. The method according to claim14, wherein said regulating comprises inhibition of one or both of theP2X₃ or P2X_(2/3) receptors.
 16. A method for treating pain in asubject, comprising administering a therapeutically effective amount ofa compound of claim 1 to said patient.
 17. The method of claim 16,wherein the pain is nociceptive, dysfunctional, idiopathic, neuropathic,somatic, central, visceral, inflammatory, or procedural pain.
 18. Themethod of claim 16, wherein the pain is caused by airway, bladder orvisceral organ dysfunction.
 19. The method of claim 16, wherein the painis a migraine, back pain, neck pain, gynecological pain, pre-labor pain,labor pain, orthopedic pain, post-stroke pain, post-surgical pain, postherpetic neuralgia, sickle cell crisis, interstitial cystitis,urological pain, dental pain, headache, wound pain, surgical pain,suturing, fracture setting pain, or pain incident to biopsy.
 20. Themethod of claim 16, wherein the pain is due to inflammation, nervecompression, or a mechanical force resulting from tissue distension as aconsequence of invasion by a tumor into a tissue.
 21. The method ofclaim 16, wherein the pain is caused by esophageal cancer, colitis,cystitis, irritable bowel syndrome or idiopathic neuropathy.
 22. Themethod of claim 17, wherein the somatic pain comprises pain from bone,joint, muscle, skin or connective tissue.
 23. The method of claim 17,wherein the central pain comprises pain from brain trauma, stroke orspinal cord injury.
 24. The method of claim 17, wherein the visceralpain comprises pain from the respiratory tract, gastrointestinal tract,pancreas, urinary tract or reproductive organs.
 25. The method of claim17, wherein the dysfunctional pain comprises pain from a rheumatologiccondition, tension type headache, irritable bowel disorder orerythermalgia.
 26. The method of claim 17, wherein the nociceptive paincomprises pain from a cut, bruise, bone fracture, crush injury, burn,trauma, surgery, labor, sprain, bump, injection, dental procedure, skinbiopsy or obstruction.
 27. The method of claim 17, wherein theneuropathic pain comprises pain due to trauma, surgery, herniation of anintervertebral disk, spinal cord injury, diabetes, infection with herpeszoster, HIV/AIDS, late-stage cancer, amputation, carpal tunnel syndrome,chronic alcohol use, exposure to radiation, or an unintended side-effectof a neurotoxic treatment agent.
 28. The method of claim 17, wherein theinflammatory pain comprises pain due to joint injury, muscle injury,tendon injury, surgical procedures, infection or arthritis.
 29. Themethod of claim 17, wherein the procedural pain comprises pain from amedical, dental or surgical procedure.
 30. The method of claim 29,wherein the procedural pain is postoperative pain, associated with aninjection, draining an abscess, surgery, dermatological, dentalprocedure, ophthalmic procedure, arthroscopy or cosmetic surgery. 31.The method of claim 16, wherein the pain is caused by cancer.
 32. Themethod of claim 31, wherein the cancer is bone cancer.
 33. The method ofclaim 16, wherein the administration is oral, intramuscular, rectal,cutaneous, subcutaneous, topical, transdermal, sublingual, nasal,vaginal, epidural, intrathecal, intravesical or ocular.
 34. A method fortreating a respiratory dysfunction in a subject in need thereof, saidmethod comprising administering a therapeutically effective amount of acompound of claim 1 to the subject.
 35. The method of claim 34, whereinthe respiratory dysfunction is one or more of bronchial hyperactivity,bronchoconstriction, bronchospasm, hypersecretion, cough, coughhypersensitivity syndrome, wheezing, dyspnea, breathlessness and chesttightness.
 36. The method of claim 34, wherein the respiratorydysfunction is caused by idiopathic pulmonary fibrosis (IPF), chronicobstructive pulmonary disease (COPD), asthma, upper respiratoryinfection, interstitial lung disease (ILD), post-nasal drip, bronchitis,gastroesophageal reflux disease (GERD), treatment with an ACE(Angiotensin Converting Enzyme) inhibitor or smoking.
 37. The method ofclaim 35, wherein the cough is acute cough, sub-acute cough, chroniccough, pathologic cough, or the urge to cough.
 38. The method of claim35, wherein the cough is caused by idiopathic pulmonary fibrosis (IPF),chronic obstructive pulmonary disease (COPD), asthma, upper respiratoryinfection, interstitial lung disease (ILD), post-nasal drip, bronchitis,gastroesophageal reflux disease (GERD), treatment with an ACE(Angiotensin Converting Enzyme) inhibitor or smoking.
 39. The method ofclaim 34, wherein the administration is oral, intramuscular, rectal,cutaneous, subcutaneous, topical, transdermal, sublingual, nasal,vaginal, epidural, intrathecal, intravesical, ocular or inhalation.